WO2021073490A1 - 一种检测ctDNA中肿瘤特异基因的变异和甲基化的方法 - Google Patents

一种检测ctDNA中肿瘤特异基因的变异和甲基化的方法 Download PDF

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WO2021073490A1
WO2021073490A1 PCT/CN2020/120560 CN2020120560W WO2021073490A1 WO 2021073490 A1 WO2021073490 A1 WO 2021073490A1 CN 2020120560 W CN2020120560 W CN 2020120560W WO 2021073490 A1 WO2021073490 A1 WO 2021073490A1
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sequence
primer
primer set
mutation
methylation
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PCT/CN2020/120560
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English (en)
French (fr)
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焦宇辰
曲春枫
王宇婷
王沛
陈坤
宋欠欠
刘慧�
王京京
王思振
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中国医学科学院肿瘤医院
北京泛生子基因科技有限公司
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Application filed by 中国医学科学院肿瘤医院, 北京泛生子基因科技有限公司 filed Critical 中国医学科学院肿瘤医院
Priority to JP2022521219A priority Critical patent/JP2022551688A/ja
Priority to EP20876690.7A priority patent/EP4023795A4/en
Priority to KR1020227016270A priority patent/KR20220088724A/ko
Priority to US17/768,891 priority patent/US20230272475A1/en
Publication of WO2021073490A1 publication Critical patent/WO2021073490A1/zh

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Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a method for detecting mutation and methylation of tumor-specific genes in ctDNA.
  • Circulating tumor DNA is derived from the DNA fragments produced by apoptosis, necrosis or secretion of tumor cells, and contains the same gene mutations and apparent modifications as tumor tissue DNA, such as point mutations, gene rearrangements, fusions, Copy number variation, methylation modification, etc.
  • the detection of ctDNA can be used in various aspects such as early cancer screening, diagnosis and staging, guidance of targeted medication, efficacy evaluation, and recurrence monitoring. Combining the mutation and methylation information of tumor-specific genes carried by ctDNA can help improve the sensitivity and specificity of detection, and find cancer traces earlier, which is of great significance for early tumor screening.
  • PCR-based hot-spot mutation detection methods usually detecting one or several Hotspot mutations or known mutations, unable to detect complex mutations such as gene fusion, unable to detect unknown mutations, and small coverage
  • Capture sequencing method suitable for multiple target detection, including complex mutations, but capture kits are generally expensive and complicated to operate , It takes a long time. In the application process, it is necessary to select a suitable detection method according to the number and characteristics of the target.
  • ctDNA methylation markers are distributed in clusters, which are more specific than gene mutations, have tissue specificity, can trace the source of tumors, and have a larger number of markers, which can achieve higher sensitivity.
  • the detection methods include: 1) Methylation PCR, due to the loss of DNA and reduced sequence diversity caused by the bisulfite conversion step, this method is difficult to achieve multiple target detection; 2) Methylation capture based on probe hybridization: covering 8%-13 % CpG sites, while detecting a large number of markers, but limited by the limited amount of ctDNA, and after bisulfite treatment, the richness of the genome sequence decreases, and the probe specificity is not easy to guarantee; 3) Based on MspI enzyme Cut RRBS (Reduced Representation Bisulfite Sequencing, RRBS), the CpG site it covers is determined by the restriction site "CCGG", accounting for about 8%-10% of CpG sites, for the recognition of methylated C bases It also relies on bisulfit
  • the methylation sites detected by RRBS are concentrated in CpG islands and promoter regions, which is relatively low cost.
  • the above three methods have limited methylation PCR coverage sites; methylation capture can cover more sites, which is more stable than RRBS data; RRBS has the lowest cost and can also cover a large number of methylation sites, which is required in the application process The choice is made according to the number and characteristics of the target.
  • Mutation detection and methylation detection will cause more DNA loss during processing; 2) the bisulfite conversion step of methylation detection technology, The DNA sequence will not be able to display most of the mutation information, and the loss of this part of the DNA carrying information may lead to a decrease in the sensitivity of low-frequency mutation detection; 3) In clinical testing, it is often necessary to determine the target and plan of subsequent testing based on the results of the first test. It is necessary to draw blood again in the follow-up test, and the testing cycle is prolonged.
  • ctDNA-related clinical testing or research often needs to compare the advantages and disadvantages of multiple technologies, which requires samples that are several times the normal volume of blood drawn, which is usually unacceptable to patients; 4) Whether it is the PCR method or the capture method, the noise mutations generated during the amplification process will seriously interfere with the detection of ctDNA low-frequency mutations, causing false positive results and misleading the diagnosis and treatment of patients; 5) The content of ctDNA mutations is low, and the operation process is easy Contamination occurs, causing false positive results.
  • the purpose of the present invention is to simultaneously detect the mutation and/or methylation of multiple tumor-specific genes in ctDNA.
  • the present invention first protects a method for constructing a sequencing library, which may sequentially include the following steps:
  • step (3) Connect the DNA sample processed in step (2) to the adaptor in the adaptor mixture, and obtain a library after PCR amplification;
  • the joint mixture is composed of n joints;
  • Each adapter is obtained by forming a partial double-stranded structure from an upstream primer A and a downstream primer A;
  • the upstream primer A has a sequencing adapter, a random tag, an anchor sequence A, and a base T located at the end (such as the 3'end)
  • the downstream primer A has an anchor sequence B and a sequencing adapter B;
  • the partial double-stranded structure is formed by the reverse complementation of the anchor sequence A and the anchor sequence B;
  • the sequencing adaptor A and sequencing adaptor B are for selecting corresponding sequencing adaptors according to different sequencing platforms
  • the random tag is a random base of 8-14 bp (such as 8-10 bp, 10-14 bp, 8 bp, 10 bp or 14 bp);
  • the length of the anchor sequence A is 12-20 bp (such as 12-16 bp, 16-20 bp, 12 bp, 16 bp or 20 bp), and the number of consecutively repeated bases is ⁇ 3;
  • n linkers use n different anchor sequences, and the four bases in each anchor sequence are balanced, and the number of mismatched bases is ⁇ 3;
  • n is any natural number ⁇ 8.
  • the linker used for library construction is formed by annealing two sequences and has a "Y"-shaped structure.
  • the complementary pairing between the two sequences ie anchor sequence A and anchor sequence B
  • anchor sequence can be used as a built-in tag for sequence fixation to label the original template molecule.
  • the anchor sequence does not interact with other parts of the primer (such as forming a hairpin structure, dimer, etc.).
  • the upstream primer A may include a sequencing adapter A, a random tag, an anchor sequence A, and a base T in sequence from the 5'end.
  • the upstream primer A may be composed of a sequencing adapter A, a random tag, an anchor sequence A, and a base T in sequence from the 5'end.
  • the downstream primer A may include an anchor sequence B and a sequencing adapter B in sequence from the 5'end.
  • the downstream primer A can be composed of an anchor sequence B and a sequencing adapter B sequentially from the 5'end.
  • the "four bases in each anchor sequence A are balanced", that is, A, T, C, and G are evenly distributed.
  • the "number of mismatch bases ⁇ 3" may mean that the linker mixture contains n anchor sequence nails, and the bases between each anchor sequence nail are at least 3 different. The difference may be a different position or a different order.
  • the DNA sample may be genomic DNA, cDNA, ct DNA or cf DNA sample.
  • n may be 12.
  • the random tag may specifically be a random base of 8 bp.
  • the length of the anchor sequence A may specifically be 12 bp.
  • the nucleotide sequence of the anchor sequence A may specifically be sequence 1 from the 30th-41th position of the 5'end in the sequence listing, and sequence 3 from the 30th-41th position of the 5'end of the sequence listing respectively.
  • Sequence 5 of the sequence listing is from the 30th-41th position of the 5'end
  • the sequence of the sequence listing 7 is from the 30th-41th position of the 5'end
  • the sequence of the sequence listing 9 is from the 30th-41th position of the 5'end
  • the sequence of the sequence listing 11 From the 30-41 position of the 5'end
  • the sequence 13 from the 5'end 30-41 the sequence 15 from the 5'end 30-41
  • Sequence 19 of the Sequence Listing is from the 30-41 of the 5'end
  • Sequence 21 of the Sequence Listing is from the 30-41 of the 5'end
  • Sequence 23 of the Sequence Listing is from the 30-41 of the 5'end Shown.
  • the sequencing adapter A may specifically be a sequencing adapter of the Truseq sequencing kit from Illumina.
  • the sequencing adaptor A can be specifically shown as sequence 1 in the sequence table from positions 1-29 from the 5'end.
  • the sequencing adapter B may specifically be a sequencing adapter of the nextera sequencing kit from Illumina.
  • the sequencing linker B can be specifically as shown in sequence 2 from the 5'end 13-41 of the sequence list.
  • the linker 1 can be obtained from the single-stranded DNA molecule shown in sequence 1 of the sequence listing and the single-stranded DNA molecule shown in sequence 2 forming a partial double-stranded structure; the linker 2 can be obtained from the single-stranded DNA molecule shown in sequence 3 and sequence 4 of the sequence listing.
  • the single-stranded DNA molecule shown forms a partial double-stranded structure;
  • the linker 3 can be obtained from the single-stranded DNA molecule shown in sequence 5 of the sequence listing and the single-stranded DNA molecule shown in sequence 6 to form a partial double-stranded structure;
  • the linker 4 can be obtained from the sequence The single-stranded DNA molecule shown in sequence 7 of the list and the single-stranded DNA molecule shown in sequence 8 form a partial double-stranded structure;
  • the linker 5 can be obtained from the single-stranded DNA molecule shown in sequence 9 of the sequence listing and the single-stranded DNA molecule shown in sequence 10
  • the stranded DNA molecule forms a partial double-stranded structure;
  • the linker 6 can be obtained from the single-stranded DNA molecule shown in sequence 11 in the sequence listing and the single-stranded DNA molecule shown in sequence 12 to form a partially double-stranded structure;
  • the linker 7 can be obtained from sequence 13
  • the single-stranded DNA molecule and the single-stranded DNA molecule shown in sequence 20 form a partial double-stranded structure;
  • the linker 11 can form a partial double-stranded structure from the single-stranded DNA molecule shown in sequence 21 of the sequence listing and the single-stranded DNA molecule shown in sequence 22 Obtained;
  • the linker 12 can be obtained by forming a partial double-stranded structure between the single-stranded DNA molecule shown in sequence 23 of the sequence listing and the single-stranded DNA molecule shown in sequence 24.
  • the adaptor can be obtained by annealing the upstream primer A and the downstream primer A.
  • each joint can be mixed in an equimolar manner.
  • the method may further include the step of amplifying the library obtained in step (3).
  • the amplified primers are designed according to the adapter sequence, that is, the amplified primer must have at least a sequence that is completely consistent with a certain sequence of the adapter.
  • the primer pair used in the amplification may specifically be composed of two single-stranded DNA molecules shown in sequence 25 and sequence 26 in the sequence listing.
  • the single-stranded DNA molecule shown in sequence 25 of the sequence listing is the sequence adaptor A from the 1st to the 19th position from the 5'end.
  • the single-stranded DNA molecule shown in sequence 26 of the sequence listing is the sequencing linker B from positions 1 to 22 from the 3'end.
  • the present invention also protects the DNA library constructed by the method described above.
  • the present invention also protects a kit for constructing a sequencing library, which may include any one of the linker mixtures described above and a methylation-sensitive restriction endonuclease.
  • the kit for constructing a sequencing library can specifically be composed of any one of the aforementioned linker mixtures and methylation-sensitive restriction endonucleases.
  • the present invention also protects a kit for detecting tumor mutation and/or methylation in a DNA sample, which includes any of the above-mentioned adapter mixtures and primer combinations; the primer combination includes primer set I, primer set II, and primers Set III, primer set IV, primer set V, primer set VI, primer set VII and primer set VIII;
  • Each primer in the primer set I and the primer set II is a specific primer designed according to the region related to tumor mutation, and its role is to locate at a specific position in the genome to achieve PCR enrichment of the target region; the primer set I And the primer set II are respectively used to detect the mutation sites of the positive and negative strands of DNA;
  • Each primer in the primer set III and the primer set IV is a specific primer designed according to the tumor-specific hypermethylated region, and its function is to locate at a specific position in the genome to achieve PCR enrichment of the target region; the primer set III and The primer set IV is used to detect the methylation sites of the positive and negative strands of DNA, respectively;
  • Each of the primers in the primer set V, the primer set VI, the primer set VII and the primer set VIII includes a linker sequence and a specific sequence, and the specific sequence is used for further enrichment of the target region;
  • the two primers designed for the same mutation site are in a "nested" relationship;
  • the two primers designed for the same mutation site are in a "nested" relationship;
  • the two primers designed for the same methylation site are in a "nested" relationship;
  • the two primers designed for the same methylation site are in a "nested" relationship.
  • the "specific primers designed according to the region related to tumor mutations” can specifically be designed according to the regions of tumor-specific gene mutations (such as point mutations, indel mutations, HBV integration and other mutation forms), to design corresponding gene-specific primers .
  • the "specific primer designed according to the tumor-specific hypermethylated region” can specifically be designed according to the tumor-specific methylated region, and the corresponding gene-specific primer is designed.
  • the tumor may be a liver malignant tumor, that is, hepatocellular carcinoma.
  • the regions related to hepatocellular carcinoma mutations may specifically be related regions of hepatocellular carcinoma high-frequency mutation genes (TP53, CTNNB1, AXIN1, TERT) and hot spots for HBV integration.
  • the primer set I includes 78 single-stranded DNA molecules, and the nucleotide sequences of the 78 single-stranded DNA molecules are shown in sequence 28 to 105 in the sequence listing.
  • the primer set II includes 82 single-stranded DNA molecules, and the nucleotide sequences of the 82 single-stranded DNA molecules are shown in sequence 106 to sequence 187 in the sequence list.
  • the primer set III includes 14 single-stranded DNA molecules, and the nucleotide sequences of the 14 single-stranded DNA molecules are as shown in sequence 188 to sequence 201 in the sequence list.
  • the primer set IV includes 15 single-stranded DNA molecules, and the nucleotide sequences of the 15 single-stranded DNA molecules are shown in sequence 202 to sequence 216 in the sequence list.
  • the primer set V includes 75 single-stranded DNA molecules, and the 75 single-stranded DNA molecules sequentially include the nucleotide sequences shown in sequence 220 to sequence 294 from the 5'end to the 3'end.
  • the primer set VI includes 79 single-stranded DNA molecules, and the 79 single-stranded DNA molecules sequentially include the nucleotide sequences shown in sequence 295 to sequence 373 from the 5'end to the 3'end.
  • the primer set VII includes 14 single-stranded DNA molecules, and the 14 single-stranded DNA molecules sequentially include the nucleotide sequences shown in sequence 374 to sequence 387 from the 5'end to the 3'end.
  • the primer set VIII includes 15 single-stranded DNA molecules, and the 15 single-stranded DNA molecules sequentially include the nucleotide sequences shown in sequence 388 to sequence 402 from the 5'end to the 3'end.
  • the nucleotide sequences of the 75 single-stranded DNA molecules in the primer set V can be as shown in sequence 220 to sequence 294 in the sequence listing.
  • the nucleotide sequences of the 79 single-stranded DNA molecules in the primer set VI can be as shown in sequence 295 to sequence 373 in the sequence listing.
  • the nucleotide sequences of the 14 single-stranded DNA molecules in the primer set VII can be as shown in sequence 374 to sequence 387 in the sequence listing.
  • the nucleotide sequences of the 15 single-stranded DNA molecules in the primer set VIII can be as shown in sequence 388 to sequence 402 in the sequence listing.
  • the primer set I can specifically consist of the 78 single-stranded DNA molecules.
  • the primer set II can specifically be composed of the 82 single-stranded DNA molecules.
  • the primer set III can specifically be composed of the 14 single-stranded DNA molecules.
  • the primer set IV can specifically be composed of the 15 single-stranded DNA molecules.
  • the primer set V can specifically be composed of the 75 single-stranded DNA molecules.
  • the primer set VI can specifically consist of the 79 single-stranded DNA molecules.
  • the primer set VII can specifically be composed of the 14 single-stranded DNA molecules.
  • the primer set VIII can be specifically composed of the 15 single-stranded DNA molecules.
  • any one of the aforementioned kits can specifically be composed of any one of the aforementioned linker mixtures and the aforementioned primer combination.
  • any one of the aforementioned primer combinations can be specifically composed of the primer set I, the primer set II, the primer set III, the primer set IV, the primer set V, the primer set VI, and the primer set VII And the primer set VIII.
  • kits may also include reagents for DNA extraction, reagents for DNA library construction, reagents for library purification, reagents for library capture, and other materials for library construction.
  • the present invention also protects any of the aforementioned primer combinations.
  • the primer combination can be used to detect tumor mutation and/or methylation in a DNA sample.
  • the present invention also protects S1) or S2) or S3):
  • the tumor may be a liver malignant tumor, that is, hepatocellular carcinoma.
  • the present invention also protects a method for detecting target mutation and/or methylation in a DNA sample, which may include the following steps:
  • step (2) Perform two rounds of nested PCR amplification on the library obtained in step (1), sequence the product, and analyze the occurrence of target mutation and/or methylation in the DNA sample according to the sequencing result;
  • the first round of PCR amplification is performed using the primer combination A;
  • Primer combination A is composed of upstream primer A and downstream primer combination A;
  • the upstream primer A is a library amplification primer used for library amplification in step (1);
  • the downstream primer combination A is a combination of Y primers designed according to X target points; X and Y are both natural numbers greater than 1, and X ⁇ Y;
  • the second round of PCR amplification is carried out with primer combination B;
  • Primer combination B is composed of upstream primer B, downstream primer combination B and index primer;
  • the upstream primer B is a library amplification primer and the 3'end is the same as the upstream primer A, and is used for the amplification of the product of the first round of PCR;
  • the index primer includes a segment A for sequencing, an index sequence for distinguishing samples, and a segment B for sequencing from the 5'end;
  • the primer in the downstream primer combination B has the segment B and forms a nested relationship with the primer in the downstream primer combination A that detects the same target point.
  • the nucleotide sequence of the upstream primer B may be as shown in sequence 217 in the sequence listing.
  • the index primer can be specifically composed of the segment A, the index sequence and the segment B from the 5'end.
  • the nucleotide sequence of the segment A may be as shown in sequence 218 in the sequence listing.
  • the nucleotide sequence of the segment B may be as shown in sequence 219 in the sequence listing.
  • the partial sequence of the upstream primer A is exactly the same as the sequence of the "sequencing adapter A of the upstream primer A of each linker".
  • the upstream primer B is used to complement the linker sequence of the full library molecule, so that the amplified product can be directly sequenced.
  • the partial nucleotide sequence of the upstream primer B and the upstream primer A are completely identical.
  • the nucleotide sequence of the upstream primer A may specifically be as shown in sequence 27 in the sequence listing.
  • the nucleotide sequence of the upstream primer B can be specifically as shown in sequence 188 in the sequence listing.
  • the downstream primer set A is composed of any one of the above-mentioned primer set I and primer set II.
  • the downstream primer set B is composed of any one of the above-mentioned primer set V and primer set VI.
  • the product amplified using primer set I was used as the template for the second round of amplification and was amplified using primer set V.
  • the product amplified using primer set II was used as the template for the second round of amplification and was amplified using primer set VI. Finally, the amplified products are mixed in equal volumes.
  • the downstream primer combination A is composed of any one of the aforementioned primer group III and primer group IV.
  • the downstream primer set B is composed of any one of the above-mentioned primer set VII and primer set VIII.
  • primer set III and primer set IV to perform the first round of PCR amplification on the template respectively.
  • the product amplified using primer set III was used as the template for the second round of amplification and was amplified using primer set VII.
  • the product amplified using primer set IV was used as the template for the second round of amplification and was amplified using primer set VIII. Finally, the amplified products are mixed in equal volumes.
  • the method for analyzing target mutations in the DNA sample may be: backtracking the DNA molecules whose sequencing data meets criterion A to a molecular cluster; mark the molecular clusters satisfying criterion B as a pair of duplex molecular clusters; For mutations, if the following (a1) or (a2) are met, the mutation is a true mutation from the original DNA sample: (a1) At least a pair of duplex molecular clusters are supported (this condition only supports the capture of sequencing data, for race The data of is not applicable); (a2) At least 4 molecular clusters are supported; Standard A meets 1, 2 and 3 at the same time; 1The length of the DNA insert is the same and the sequence is the same except for the mutation site; 2The random tag sequence is the same; 3anchor The sequence is the same; Standard B meets both 4 and 5; 4The length of the DNA insert is the same and the sequence is the same except for the mutation site; 5 The anchor sequence at both ends of the molecular cluster is the same but the position is
  • the above-mentioned DNA inserts specifically refer to amplified DNA fragments other than adaptors.
  • the present invention also protects a method for detecting multiple target mutations and/or methylation in a DNA sample, which may include the following steps:
  • step (2) Perform target region enrichment and sequencing on the library of step (1), and analyze the occurrence of target mutations and/or methylation in the DNA sample according to the sequencing results.
  • the method for analyzing target mutations in the DNA sample may be: backtracking the DNA molecules whose sequencing data meets criterion A to a molecular cluster; mark the molecular clusters satisfying criterion B as a pair of duplex molecular clusters; For mutations, if the following (a1) or (a2) are met, the mutation is a true mutation from the original DNA sample: (a1) at least a pair of duplex molecular clusters are supported; (a2) at least 4 molecular clusters are supported; Standard A meets 1, 2 and 3 at the same time; 1DNA insert length is the same and the sequence is the same except for the mutation site; 2The random tag sequence is the same; 3The anchor sequence is the same; Standard B meets both 4 and 5; 4DNA insert length Same and the same sequence except the mutation site; 5 The anchor sequence at both ends of the molecular cluster is the same but the position is opposite.
  • the target region enrichment can be performed by using an existing commercially available targeted capture kit (for example, Agilent sureselect XT targeted capture kit, Agilent 5190-8646), and the primer pair in the last step of PCR amplification is replaced by primer A.
  • a primer pair consisting of primer B.
  • the nucleotide sequence of the primer A may be as shown in sequence 403 in the sequence listing.
  • the primer B may include segment A, index sequence and segment B.
  • the primer B may specifically consist of the segment A, the index sequence and the segment B.
  • the nucleotide sequence of the segment A may be as shown in sequence 404 in the sequence listing.
  • the nucleotide sequence of the segment B may be as shown in sequence 405 in the sequence listing.
  • the target mutation and/or methylation may be tumor mutation and/or methylation.
  • the tumor may be a malignant tumor of the liver, that is, hepatocellular carcinoma.
  • the DNA sample is digested with a methylation-sensitive restriction endonuclease to form a DNA fragment (at this time, both ends of the DNA fragment form sticky ends, and the nucleotide sequence of the single-stranded part of the end is the breakpoint sequence ); After the DNA fragment is repaired, it is connected to the adaptor (5' end and 3'end are each connected to a adaptor, which may be the same or opposite). For the DNA molecule at this time, the difference between the two adaptors The DNA fragment is the DNA insert.
  • the present invention provides a method that can simultaneously detect tumor-specific gene mutations (including point mutations, indel mutations, HBV integration and other mutation forms) and/or methylation in ctDNA in a sample.
  • the quantity requirement is low, and the MC library prepared by this method can support 10-20 follow-up tests.
  • the result of each test can represent the mutation status of all the original ctDNA specimens and the methylation modification of the area covered by the restriction site. And will not cause a decrease in sensitivity and specificity.
  • the library constructed using this method can be used for both PCR hot spot detection and capture method sequencing.
  • the added DNA barcode can effectively filter out false positive results and achieve high-specificity sequencing based on duplex.
  • the library construction method is not only suitable for cfDNA samples, but also for genomic DNA or cDNA samples.
  • the invention has important clinical significance for early tumor screening, disease tracking, curative effect evaluation, prognosis prediction, etc., and has great application value.
  • Figure 1 is a schematic diagram of the adapter and primer architecture.
  • Figure 2 is a schematic diagram of RaceSeq target region enrichment and library construction.
  • Figure 3 is a schematic diagram of MC library capture and duplex sequencing.
  • Figure 4 shows the results of detecting the methylation level of the AK055957 gene by the Padlock method and the mutation/methylation co-detection method (that is, the method provided by the present invention).
  • Figure 5 shows the results of mutation and mutation frequency detection by the mutation detection method alone and the mutation/methylation combined detection method.
  • test materials used in the following examples are all purchased from conventional biochemical reagent stores.
  • the TE buffer in the following examples is a product of ThermoFisher, and the product catalog number is 12090015.
  • Restriction Enzyme and Restriction Enzyme 10 ⁇ Buffer are products of ThermoFisher. Restriction Enzyme and Restriction Enzyme 10 ⁇ Buffer can be selected according to different target regions to be tested. The selection criterion is that there is at least one restriction site for the methylation-sensitive restriction enzyme in the tested region.
  • the digested product obtained in step 1 is purified and enriched using allelic MiniMax TM high-efficiency free DNA enrichment and separation kit (standard version) (product of Eck, catalog number A17622-50) to obtain a purified product.
  • allemicMax TM high-efficiency free DNA enrichment and separation kit standard version
  • step two Take the purified product obtained in step two, configure the reaction system as shown in Table 3, and then perform end repair on the PCR machine and add A at the 3'end according to the reaction procedure in Table 4 to obtain the reaction product (stored at 4°C).
  • reaction product is connected to the adapter
  • the single-stranded DNA molecules in Table 6 were dissolved in TE buffer and diluted to a concentration of 100 ⁇ M. Mix two single-stranded DNA molecules in the same group in equal volumes (50 ⁇ l each), and then perform annealing (annealing procedure: 95°C, 15min; 25°C, 2h) to obtain 12 groups of DNA solutions.
  • the 12 groups of DNA solutions are of equal volume Mix, get Adapter Mix.
  • 8 Ns represent random tags of 8bp. In practical applications, the length of the random tag can be 8-14bp.
  • the underline indicates the 12bp anchor sequence.
  • the underlined part is reverse complementary. Annealing allows upstream and downstream sequences to join together to form a linker.
  • the anchor sequence can be used as a built-in tag for sequence fixation to label the original template molecule.
  • the anchor sequence length can be 12-20 bp, the number of consecutive repeating bases is not more than 3, and it cannot interact with other parts of the primer (such as forming a hairpin structure, dimer, etc.), 12 groups of bases at each position Balanced (that is, A, T, C, and G are evenly distributed), and the number of mismatched bases is ⁇ 3 (that is, there are at least 3 different bases between each anchor sequence; the difference can be a different position or a different order).
  • the bold T at the end of the upstream sequence is complementary to the "A" at the end of the original molecule, and TA is connected.
  • positions 1 to 21 from the 5'end are the sequencing primer binding sequences, and positions 1 to 19 from the 5'end are the library amplification primers.
  • the non-underlined part (nextera sequencing kit from Illumina) is the sequencing primer binding sequence, and positions 1 to 22 from the 3'end are the part of the library amplification primer.
  • connection product The structure of the connection product is shown in Figure 1. Among them, a is the adapter part, b and f are library amplification primers, c is an 8bp random tag (indicated by 8 N in Table 6), d is a 12bp anchor sequence (indicated by the underline in Table 6), and e is Insert fragment (cfDNA).
  • step 5 Take the PCR template obtained in step 5, configure the reaction system according to Table 7, and perform PCR amplification according to Table 8 to obtain PCR amplified products (stored at 4°C).
  • MC_F (sequence 25): 5'-GACACGACGCTCTTCCGAT-3';
  • MC_R (sequence 26): 5'-GTGGGCTCGGAGATGTGTATAA-3'.
  • the MC library can support 10-20 follow-up tests, and the results of each test can represent the mutation status of all original samples and the methylation modification of the area covered by the restriction site, without causing sensitivity and specificity The reduction.
  • the library construction method is not only suitable for cfDNA samples, but also for genomic DNA or cDNA samples.
  • Example 2 RaceSeq enriches the target region and constructs a sequencing library
  • the design is designed for the relevant regions of my country's hepatocellular carcinoma high-frequency mutation genes (TP53, CTNNB1, AXIN1, TERT), HBV integration hot spots, and hepatocellular carcinoma-specific hypermethylation regions (EMX1, LRRC4, BDH1, etc.)
  • the primers of, and the fixed primers are matched to perform two rounds of PCR amplification on the MC library, and the amplified product is the sequencing library.
  • a is the upstream primer of the first round of library amplification
  • b is the upstream primer of the second round of library amplification
  • c is the downstream primer library of the first round of library amplification, used for specific target sequence enrichment
  • d is the second round of library amplification.
  • e is index primer, used to add index sequence.
  • Example 1 Take 300ng of the MC library prepared in Example 1, divide it into two parts, configure the reaction system in Table 9 (one part is added to GSP1A mix, and the other part is added to GSP1B mix), and perform the first round of PCR amplification according to the reaction program in Table 11.
  • the amplification product of the first round is obtained (a total of two amplification products of the first round are obtained, one is the amplification product of GSP1A mix, and the other is the amplification product of GSP1B mix).
  • Upstream primer 1355 (sequence 27): 5'-TCTTTCCCTACACGACGCTCTTCCGAT-3'.
  • GSP1A mix Dissolve and dilute each primer in the primer pool GSP1A in Table 10 with TE buffer to a concentration of 100 ⁇ M, then mix in equal volumes, and dilute to 0.3 ⁇ M with TE buffer.
  • the primers in the primer pool GSP1A are used to amplify the positive strand of the template.
  • GSP1B mix Dissolve and dilute each primer in the primer pool GSP1B in Table 10 with TE buffer to a concentration of 100 ⁇ M, then mix in equal volumes, and dilute to 0.3 ⁇ M with TE buffer.
  • the primers in the primer pool GSP1B are used to amplify the negative strand of the template.
  • the primers with the same number detect the same mutation site from both positive and negative directions, and use at the same time to maximize the enrichment of the original molecular information.
  • step 2 Purify the two first-round amplification products obtained in step 1 with 30-60 ⁇ l (ie 1-2 times the volume) of AMPure XP magnetic beads, and then elute with 25 ⁇ l DNase/RNase-Free Water to obtain the first Round to purify the product.
  • step 3 Using the first round of purified products obtained in step 2 as a template, configure the reaction system in Table 12 (when GSP1A mix amplification products are used as templates, use GSP2A mix amplification; when GSP1Bmix amplification products are used as templates, use GSP2B mix amplification), and perform the second round of PCR amplification according to the reaction program in Table 14 to obtain the second round of amplification products (stored at 4°C).
  • Upstream primer 3355 (sequence 217):
  • GSP2A mix Dissolve and dilute each primer in the primer pool GSP2A in Table 13 with TE buffer to a concentration of 100 ⁇ M, then mix in equal volumes, and dilute to 0.3 ⁇ M with TE buffer.
  • the primers in the primer pool GSP2A are used to amplify the positive strand of the template.
  • GSP2B mix Dissolve and dilute each primer in the primer pool GSP2B in Table 13 with TE buffer to a concentration of 100 ⁇ M, then mix in equal volumes, and dilute to 0.3 ⁇ M with TE buffer.
  • the primers in the primer pool GSP2B are used to amplify the negative strand of the template.
  • positions 1 to 15 from the 5'end are the part that binds to the Index primer.
  • the primers with the same primer number in the GSP2A mix and GSP1A mix (that is, the last four digits of the primer number are the same) are designed for the same mutation site, and the two primers form a nested relationship.
  • the primers with the same primer number in GSP2B mix and GSP2A mix (that is, the last four digits of the primer number are the same) are designed for the same mutation site, and the two primers form a nested relationship.
  • Index primer 5'-CAAGCAGAAGACGGCATACGAGAT (sequence 218)********GTGACTGGAGTTC CTTGGCACCCGAGAA -3' (sequence 219); the underlined part is the part that binds to GSP2mix.
  • ****** is the position of the index sequence, and the length of the index is 6-8bp, which is used to distinguish the sequence between samples and facilitate the mixed sequencing of multiple samples. Except for the index sequence, the rest are fixed sequences from Illumina's small RNA sequencing kit.
  • NA no primer
  • step 4 for the second round of amplification using GSP2A mix and the product of the second round of amplification using GSP1B mix are mixed in equal volumes, and purified with AMPure XP magnetic beads in a ratio of 1:(1-2) , And then eluted with 50 ⁇ l DNase/RNase-Free Water to obtain the second round of purified product, which is a sequencing library that can be sequenced on the Illumina Hiseq X platform.
  • the DNA random tag on the MC library is added to the downstream of the Read1 sequence of the sequencing library together with the cfDNA sequence.
  • DNA random tag sequence, anchor sequence, cfDNA sequence (c, d, e sequence in Figure 1) are obtained in sequence.
  • the analysis method of hepatocellular carcinoma-specific gene mutations is as follows: DNA molecules whose sequencing data meets criterion A are traced back to a molecular cluster; molecular clusters that meet criterion B are marked as a pair of duplex molecular clusters; for a certain mutation, if it meets The following (a1) or (a2), the mutation is a true mutation from the original DNA sample: (a1) at least a pair of duplex molecular clusters are supported; (a2) at least 4 molecular clusters are supported; standard A is satisfied at the same time 1 , 2 and 3; 1DNA insert length is the same and the sequence is the same except the mutation site; 2The random tag sequence is the same; 3The anchor sequence is the same; Standard B meets both 4 and 5; 4The DNA insert has the same length except the mutation site The outer sequence is the same; 5 The anchor sequence at both ends of the molecular cluster is the same but the position is opposite.
  • target region enrichment can be captured based on optimized design of existing commercial targeted capture kits.
  • capture based on methylated regions please refer to Roche SeqCap Epi CpGiant Enrichment Kit (Roche 07138881001) or Illumina Infinium Methylation EPIC BeadChipWG-317-1001.
  • the design of targeted capture of methylated regions needs to be based on the coverage of the restriction site Screen and adjust the bases converted based on bisulfite treatment in the probe.
  • For capture based on gene variant regions please refer to Agilent sureselect XT Targeted Capture Kit (Agilent5190-8646). Only the primers of the last step of PCR amplification are replaced with the following primers:
  • the upstream primer is: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCT ACACGACGCTCTTCCGAT CT-3' (sequence 403) ("a" in Figure 3), the underlined part is the same as the primer MC_F part), which functions to amplify the library, and the rest is the sequencing laboratory of the Illumina sequencing platform The required fixed sequence.
  • the downstream primer is: 5'-CAAGCAGAAGACGGCATACGAGAT (sequence 404)********GTCTC GTGGGCTCGGAGATGTGTATAA -3' (sequence 405) ("b" in Figure 3), the underlined part is the same as the primer MC_R, which acts as an extension Increase the library.
  • the position of the index sequence is 6-8bp, which is used to distinguish the sequence between samples and facilitate the mixed sequencing of multiple samples.
  • the remaining part is the fixed sequence required for sequencing on the Illumina sequencing platform.
  • the captured library and MC library have the same DNA random tag sequence, anchor sequence and cfDNA sequence, which are located downstream of Read1 in sequence.
  • the DNA molecules whose sequencing data meets the standard A are traced back to a molecular cluster; the standard A meets 1, 2 and 3 at the same time; 1the DNA insert has the same length and the sequence is the same except for the mutation site; 2the random tag sequence is the same; 3anchored sequence the same. Mark the molecular clusters that meet criterion B as a pair of duplex molecular clusters; criterion B meets both 4 and 5; 4DNA inserts are the same length and have the same sequence except for the mutation site; 5The anchor sequences at both ends of the molecular cluster are the same but the positions are opposite .
  • the mutation is a true mutation from the original DNA sample: (a1) at least a pair of duplex molecular clusters are supported; (a2) at least 4 molecules Cluster support.
  • the reliability of mutations supported by a pair of duplex molecular clusters is higher, and false positive mutations can be reduced by 90%.
  • step 2 After completing step 1, take each cfDNA sample, construct an MC library according to the method in Example 1, and then perform RaceSeq target region enrichment and sequencing according to the method in Example 2 to obtain the methylation level of the AK055957 gene.
  • Padlock is a methylation-targeted sequencing technology.
  • the Padlock probe conformation is similar to that of a padlock. It can be applied to high-throughput methylation-targeted sequencing. It is an efficient method for building libraries after bisulfite conversion. BSPP".
  • cfDNA After being converted by bisulphite, cfDNA can be amplified and ligated into a circle when paired with the capture arm of the sulfite lock probe (BSPP). Exonuclease can be used to screen out the lock probes connected to the circle. Needle, the amplified products are sequenced to obtain the corresponding DNA methylation information.
  • BSPP sulfite lock probe
  • test results are shown in Figure 4.
  • the results show that the Padlock method and the mutation/methylation co-detection method (that is, the method provided by the present invention) have basically the same detection results for the methylation level of the AK055957 gene (belonging to the hepatocellular carcinoma specific gene).
  • step (1) After completing step (1), take 5-40ngcfDNA, configure the reaction system as shown in Table 1, and then perform digestion treatment in a PCR machine to obtain the digestion product (stored at 4°C).
  • the restriction enzyme digestion time is 0h, 0.2h, 0.4h, 0.6h, 0.8h or 1h.
  • step (2) After completing step (2), take the digested product, construct an MC library according to the methods in Example 1 to 2 to 6, and then perform RaceSeq target region enrichment and sequencing according to the method in Example 2.
  • the sequencing data of DNA molecules with the same random tag sequence, the same length of the DNA insert, and the same sequence except the mutation site are traced back to a molecular cluster. If the number of molecules in the cluster is greater than 5 and the consensus rate of molecular mutations in the cluster >80% and the number of clusters is ⁇ 5, then the mutation is a true mutation from the original DNA sample. The proportion of clusters containing mutations in the molecule is the mutation frequency.
  • step (1) After completing step (1), take 5-40ng cfDNA, configure the reaction system as shown in Table 3, and then perform end repair on the PCR machine and add A at the 3'end according to the reaction program in Table 4 to obtain the reaction product ( Store at 4°C).
  • step (2) After completing step (2), take the reaction product, construct an MC library according to the method 4 to 6 in Example 1, and then perform RaceSeq target region enrichment and sequencing according to the method in Example 2.
  • the sequencing data of DNA molecules with the same random tag sequence, the same length of the DNA insert, and the same sequence except the mutation site are traced back to a molecular cluster. If the number of molecules in the cluster is greater than 5 and the consensus rate of molecular mutations in the cluster >80% and the number of clusters is ⁇ 5, then the mutation is a true mutation from the original DNA sample. The proportion of clusters containing mutations in the molecule is the mutation frequency.
  • the mutant standard product is a product of Horizon Discovery, and the product catalog number is HD701.
  • Example 1 Take the mutant standard product, construct the MC library according to the method from 1 to 6 in Example 1, and then follow the method in Example 2 (only replace GSP2A mix in step 3 with GSP2A mix-1, and GSP2B mix with GSP2B mix -1) Perform RaceSeq target region enrichment and sequencing.
  • GSP2A mix-1 Dissolve and dilute each primer in the primer pool GSP2A in Table 15 with TE buffer to a concentration of 100 ⁇ M, then mix in equal volumes, and dilute to 0.3 ⁇ M with TE buffer.
  • the primers in the primer pool GSP2A are used to amplify the positive strand of the template.
  • GSP2B mix-1 Dissolve and dilute each primer in the primer pool GSP2B in Table 15 with TE buffer to a concentration of 100 ⁇ M, then mix in equal volumes, and dilute to 0.3 ⁇ M with TE buffer.
  • the primers in the primer pool GSP2B are used to amplify the negative strand of the template.
  • GSP2A HA2123 ctccaggaagcctacgtgatg EGFR 7 55249071
  • GSP2A HA2124 acctccaccgtgcagctc EGFR 7 55259514
  • GSP2A HA2125 ccgcagcatgtcaagatcacag PIK3CA 3 178916875
  • GSP2B HB2094 ggttgaaaaagccgaaggtcac PIK3CA 3 178921551
  • GSP2B HB2095 catttgactttaccttatcaatgtctcgaa PIK3CA 3 178936082
  • GSP2B HB2096 acttacctgtgactccatagaaaatctt PIK3CA 3 178952072
  • GSP2B HB2097 caatccatttttgttgtccagcc KRAS 12 25398285
  • the test results are shown in Table 16.
  • the results show that the mutation frequency of the mutation site obtained by the mutation/methylation co-detection method for the mutation standard is basically close to the theoretical value. It can be seen that the mutation/methylation co-detection method has high accuracy in detecting the mutation of hepatocellular carcinoma specific genes (such as CTNNB1 gene, TP53 gene, AXIN1 gene).
  • geneID represents the number of the gene in the Ensemble database
  • Ref is the normal type
  • Alt is the type after gene mutation
  • INS stands for insertion
  • DEL stands for deletion
  • SNP single base mutation
  • the human methylation and non-methylation standards are the products of Zymo Research, and the product catalog number is D5014.
  • the proportion of methylation standards is 0%, 20% or 100%, that is, tumor-specific genes (BDH1 gene, EMX1 gene, LRRC4 gene, CLEC11A gene, HOXA1 gene, AK055957 gene, COTL1 gene, ACP1 gene Or DAB2IP gene) methylation ratio is 0%, 20% or 100%.
  • Example 2 Take the sample to be tested, construct the MC library according to the method in Example 1, and then perform the RaceSeq target region enrichment and sequencing according to the method in Example 2 to obtain the detection value of the methylation site.
  • the test results are shown in Table 17 and Table 18 (the last four bits of the sample type are the names of tumor-specific genes).
  • the mutation/methylation co-detection method is used to detect methylation standards, and the detection value is basically close to the theoretical value. It can be seen that the mutation/methylation co-detection method has the methylation level of tumor-specific genes (such as BDH1 gene, EMX1 gene, LRRC4 gene, CLEC11A gene, HOXA1 gene, AK055957 gene, COTL1 gene, ACP1 gene, DAB2IP gene) The detection has high accuracy.
  • Example 6 Application of mutation/methylation co-detection method in cfDNA of patients with hepatocellular carcinoma
  • Example 2 Take 5-40ng cfDNA, construct an MC library according to Example 1, and perform RaceSeq target region enrichment and sequencing according to the method in Example 2.
  • the mutation/methylation co-detection method can be applied to the detection of hepatocellular carcinoma cfDNA samples.
  • the invention discloses a method that can simultaneously detect the mutations (including point mutations, indel mutations, HBV integration and other mutation forms) and/or methylation of tumor-specific genes in ctDNA in a sample.
  • the quantity requirement is low, and the MC library prepared by this method can support 10-20 follow-up tests.
  • the result of each test can represent the mutation status of all the original ctDNA specimens and the methylation modification of the area covered by the restriction site. And will not cause a decrease in sensitivity and specificity.
  • the library construction method is not only suitable for cfDNA samples, but also for genomic DNA or cDNA samples.
  • the invention has important clinical significance for early tumor screening, disease tracking, curative effect evaluation, prognosis prediction, etc., and has great application value.

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Abstract

本发明公开了一种检测ctDNA中肿瘤特异基因的变异和甲基化的方法，该方法可以在一份样本中同时检测ctDNA中的肿瘤特异基因的变异(包括点突变、插入缺失突变、HBV整合等多种突变形式)和/或甲基化的方法，不仅样本量需求低，而且该方法制备的MC文库可以支持10-20次的后续检测，每次检测的结果都可以代表全部原始ctDNA标本的突变状况及酶切位点覆盖区域的甲基化修饰情况，且不会造成灵敏度和特异性的降低。本发明对肿瘤早期筛查、病情追踪、疗效评估、预后预测等具有重要的临床意义，有重大的应用价值。

Description

一种检测ctDNA中肿瘤特异基因的变异和甲基化的方法 技术领域
本发明属于生物医学领域，具体涉及一种检测ctDNA中肿瘤特异基因的变异和甲基化的方法。
背景技术
循环肿瘤DNA(circulating tumor DNA，ctDNA)来源于肿瘤细胞的凋亡、坏死或分泌产生的DNA片段，含有与肿瘤组织DNA相同的基因变异和表观修饰，如点突变、基因重排、融合、拷贝数变异、甲基化修饰等。ctDNA的检测可以应用于癌症早期筛查、诊断和分期、指导靶向用药、疗效评估、复发监测等各方面。结合ctDNA携带的肿瘤特异基因的变异和甲基化两方面信息，有助于提高检测的灵敏度和特异性，更早发现癌症踪迹，对于肿瘤早筛有重要意义。
现有的基因变异检测和甲基化检测需要遵循不同的技术路线。ctDNA基因变异的检测，由于受到ctDNA在cfDNA中占比较低的限制，实质上是低频突变的检测，现有技术分为两类：1)基于PCR的热点突变检测法，通常检测一个或几个热点突变或已知突变，无法检测基因融合等复杂突变，无法检测未知突变，覆盖范围较小；2)捕获测序法：适合多重靶标检测，包括复杂突变，但捕获试剂盒一般价格昂贵、操作复杂、耗时较长。在应用过程中，需要根据靶标的数量和特性，选取适合的检测方法。ctDNA甲基化标志物的优点在于成簇分布，与基因变异相比特异性更高，具有组织特异性，可以追溯肿瘤来源，标志物数量更多，可以达到更高的灵敏度；其检测方法包括：1)甲基化PCR，由于重亚硫酸盐转化步骤造成DNA损失及序列多样性降低，这种方法难以实现多重靶标检测；2)基于探针杂交的甲基化捕获：可覆盖8％-13％的CpG位点，同时检测大量标志物，但受到有限的ctDNA起始量的限制，并且经重亚硫酸盐处理后，基因组序列丰富度下降，探针特异性不易保证；3)基于MspI酶切的RRBS(Reduced representation bisulfite sequencing，RRBS)，它所覆盖的CpG位点由酶切位点“CCGG”决定，约占8％-10％的CpG位点，对甲基化C碱基的识别同样依赖于重亚硫酸盐转化。RRBS检测的甲基化位点集中在CpG岛和启动子区，成本较低。上述3种方法甲基化PCR覆盖位点有限；甲基化捕获可覆盖较多位点，相比RRBS数据更稳定；RRBS成本最低，也可以覆盖大量甲基化位点，在应用过程中需要根据靶标的数量和特性进行取舍。
目前没有简单、低成本和可靠的方案，实现同时检测ctDNA中的基因变异和甲基化这两种重要的肿瘤特异标志物。主要存在以下困难：1)一次抽血获得ctDNA标本量有限，通常仅够支持1-2次检测，这就造成ctDNA临床检测中通常是单平台、一次性的，难以在一份样本中同时实现突变检测和甲基化检测，尤其是依赖于重亚硫酸盐转化的甲基化检测技术，处理过程中会造成较多的DNA损失；2)甲基化检测技术的重亚硫酸盐转化步骤，将导致DNA序列无法呈现大 部分突变信息，这部分DNA携带信息的损失可能会导致低频突变检测灵敏度的降低；3)临床检测中经常需要根据首次检测的结果判断后续检测的目标和方案，这就需要在后续检测中重新抽血，检测周期延长；另外，ctDNA相关临床检测或研究中经常需要比较多种技术的优劣，这就需要数倍于正常抽血量的标本，患者通常无法接受；4)无论是PCR法还是捕获法，扩增过程中产生的噪音突变会严重干扰ctDNA低频突变的检测，造成假阳性结果，误导患者的诊断和治疗；5)ctDNA突变含量低，操作过程中易发生污染，造成假阳性结果。
发明公开
本发明的目的是同时检测ctDNA中多种肿瘤特异基因的变异和/或甲基化。
本发明首先保护一种测序文库构建方法，可依次包括如下步骤：
(1)取DNA样本，用甲基化敏感限制性内切酶酶切；
(2)将步骤(1)酶切后的DNA样本依次进行末端修复和3’端加A处理；
(3)将步骤(2)处理后的DNA样本与接头混合物中的接头连接，经过PCR扩增后得到文库；
所述接头混合物由n个接头组成；
每个接头均由一条上游引物甲和一条下游引物甲形成部分双链结构得到；上游引物甲中具有测序接头甲、随机标签、锚定序列甲和位于末端(如3’末端)的碱基T；下游引物甲中具有锚定序列乙和测序接头乙；所述部分双链结构由锚定序列甲和锚定序列乙反向互补形成；
所述测序接头甲和测序接头乙为根据不同测序平台选择对应的测序接头；
所述随机标签为8-14bp(如8-10bp、10-14bp、8bp、10bp或14bp)的随机碱基；
所述锚定序列甲长度为12-20bp(如12-16bp、16-20bp、12bp、16bp或20bp)，连续重复碱基≤3个；
n个接头采用n个不同的锚定序列甲，且每个锚定序列甲中四种碱基平衡，错配碱基数≥3；
n为≥8的任意自然数。
通常，建库用的接头由两条序列退火而成，呈“Y”型结构，两条序列间互补配对的部分(即锚定序列甲和锚定序列乙)称之为锚定序列。所述锚定序列可作为序列固定的内置标签，用于标记原始模板分子。
所述锚定序列不与引物其它部分相互作用(如形成发卡结构、二聚体等)。
所述上游引物甲自5’端依次可包括测序接头甲、随机标签、锚定序列甲和碱基T。
所述上游引物甲自5’端依次可由测序接头甲、随机标签、锚定序列甲和碱基T组成。
所述下游引物甲自5’端依次可包括锚定序列乙和测序接头乙。
所述下游引物甲自5’端依次可由锚定序列乙和测序接头乙组成。
所述“每个锚定序列甲中四种碱基平衡”即A、T、C和G均匀分布。
所述“错配碱基数≥3”可为所述接头混合物包含n个锚定序列甲，各个锚定序列甲之间的碱基至少有3个不同。该不同可为位置不同或顺序不同。
所述DNA样本可为基因组DNA、cDNA、ct DNA或cf DNA样本。
所述n具体可为12。
所述随机标签具体可为8bp的随机碱基。
所述锚定序列甲长度具体可为12bp。
当n＝12时，所述锚定序列甲的核苷酸序列具体分别可为序列表的序列1自5’端第30-41位、序列表的序列3自5’端第30-41位、序列表的序列5自5’端第30-41位、序列表的序列7自5’端第30-41位、序列表的序列9自5’端第30-41位、序列表的序列11自5’端第30-41位、序列表的序列13自5’端第30-41位、序列表的序列15自5’端第30-41位、序列表的序列17自5’端第30-41位、序列表的序列19自5’端第30-41位、序列表的序列21自5’端第30-41位、序列表的序列23自5’端第30-41位所示。
所述测序接头甲具体可为来自Illumina公司的Truseq测序试剂盒的测序接头。所述测序接头甲具体可如序列表的序列1自5’端第1-29位所示。
所述测序接头乙具体可为来自Illumina公司的nextera测序试剂盒的测序接头。所述测序接头乙具体可如序列表的序列2自5’端第13-41位所示。
当n＝12时，所述12个接头如下：
接头1可由序列表的序列1所示的单链DNA分子和序列2所示的单链DNA分子形成部分双链结构得到；接头2可由序列表的序列3所示的单链DNA分子和序列4所示的单链DNA分子形成部分双链结构得到；接头3可由序列表的序列5所示的单链DNA分子和序列6所示的单链DNA分子形成部分双链结构得到；接头4可由序列表的序列7所示的单链DNA分子和序列8所示的单链DNA分子形成部分双链结构得到；接头5可由序列表的序列9所示的单链DNA分子和序列10所示的单链DNA分子形成部分双链结构得到；接头6可由序列表的序列11所示的单链DNA分子和序列12所示的单链DNA分子形成部分双链结构得到；接头7可由序列表的序列13所示的单链DNA分子和序列14所示的单链DNA分子形成部分双链结构得到；接头8可由序列表的序列15所示的单链DNA分子和序列16所示的单链DNA分子形成部分双链结构得到；接头9可由序列表的序列17所示的单链DNA分子和序列18所示的单链DNA分子形成部分双链结构得到；接头10可由序列表的序列19所示的单链DNA分子和序列20所示的单链DNA分子形成部分双链结构得到；接头11可由序列表的序列21所示的单链DNA分子和序列22所示的单链DNA分子形成部分双链结构得到；接头12可由序列表的序列23所示的单链DNA分子和序列24所示的单链DNA分子形成部分双链结构得到。
所述接头可以通过将上游引物甲和下游引物甲进行退火得到。
所述接头混合物中，每个接头可等摩尔混合。
所述方法还可包括对步骤(3)得到的文库进行扩增的步骤。所述扩增的引物是根据接头序列设计的，即所述扩增的引物至少要有一段序列与接头的某段序列完全一致。所述扩增采用的引物对具体可由序列表的序列25和序列26所示的两条单链DNA分子组成。
序列表的序列25所示的单链DNA分子即为测序接头甲自5’端第1至19位。
序列表的序列26所示的单链DNA分子即为测序接头乙自3’端第1至22位。
本发明还保护以上所述的方法构建得到的DNA文库。
本发明还保护一种用于构建测序文库的试剂盒，可包括上述任一所述的接头混合物和甲基化敏感限制性内切酶。
所述用于构建测序文库的试剂盒具体可由上述任一所述的接头混合物和甲基化敏感限制性内切酶组成。
本发明还保护一种用于检测DNA样本中肿瘤突变和/或甲基化的试剂盒，包括上述任一所述接头混合物和引物组合；所述引物组合包括引物组Ⅰ、引物组Ⅱ、引物组Ⅲ、引物组Ⅳ、引物组Ⅴ、引物组Ⅵ、引物组Ⅶ和引物组Ⅷ；
所述引物组Ⅰ和所述引物组Ⅱ中的各个引物是根据与肿瘤突变相关的区域设计的特异性引物，作用是定位于基因组特定位置，实现目标区域的PCR富集；所述引物组Ⅰ和所述引物组Ⅱ分别用于检测DNA正链和负链的突变位点；
所述引物组Ⅲ和所述引物组Ⅳ中的各个引物是根据肿瘤特异高甲基化区域设计的特异性引物，作用是定位于基因组特定位置，实现目标区域的PCR富集；所述引物组Ⅲ和所述引物组Ⅳ分别用于检测DNA正链和负链的甲基化位点；
所述引物组Ⅴ、所述引物组Ⅵ、所述引物组Ⅶ和所述引物组Ⅷ中的各个引物均包括接头序列和特异序列，特异序列用于目标区域进行进一步富集；
所述引物组Ⅴ和所述引物组Ⅰ中，针对同一突变位点设计的两个引物为“巢式”关系；
所述引物组Ⅵ和所述引物组Ⅱ中，针对同一突变位点设计的两个引物为“巢式”关系；
所述引物组Ⅶ和所述引物组Ⅲ中，针对同一甲基化位点设计的两个引物为“巢式”关系；
所述引物组Ⅷ和所述引物组Ⅳ中，针对同一甲基化位点设计的两个引物为“巢式”关系。
所述“根据与肿瘤突变相关的区域设计的特异性引物”具体可为根据肿瘤特异基因变异(如点突变、插入缺失突变、HBV整合等多种突变形式)的区域，设计对应的基因特异引物。
所述“根据肿瘤特异高甲基化区域设计的特异性引物”具体可为根据肿瘤特异甲基化区域，设计对应的基因特异引物。
所述试剂盒中，所述肿瘤可为肝脏恶性肿瘤，即肝细胞癌。
所述与肝细胞癌突变相关的区域具体可为肝细胞癌高频突变基因(TP53、CTNNB1、AXIN1、TERT)的相关区域和针对HBV整合热点区域。
上述任一所述试剂盒中，所述引物组Ⅰ包括78个单链DNA分子，78个单链DNA分子的核苷酸序列依次如序列表的序列28至序列105所示。所述引物组Ⅱ包括82个单链DNA分子，82个单链DNA分子的核苷酸序列依次如序列表的序列106至序列187所示。所述引物组Ⅲ包括14个单链DNA分子，14个单链DNA分子的核苷酸序列依次如序列表的序列188至序列201所示。所述引物组Ⅳ包括15个单链DNA分子，15个单链DNA分子的核苷酸序列依次如序列表的序列202至序列216所示。所述引物组Ⅴ包括75个单链DNA分子，75个单链DNA分子依次包括如序列表的序列220至序列294自5’末端起第16位至3’末端所示的核苷酸序列。所述引物组Ⅵ包括79个单链DNA分子，79个单链DNA分子依次包括如序列表的序列295至序列373自5’末端起第16位至3’末端所示的核苷酸序列。所述引物组Ⅶ包括14个单链DNA分子，14个单链DNA分子依次包括如序列表的序列374至序列387自5’末端起第16位至3’末端所示的核苷酸序列。所述引物组Ⅷ包括15个单链DNA分子，15个单链DNA分子依次包括如序列表的序列388至序列402自5’末端起第16位至3’末端所示的核苷酸序列。
所述引物组Ⅴ中75个单链DNA分子的核苷酸序列依次可如序列表的序列220至序列294所示。所述引物组Ⅵ中79个单链DNA分子的核苷酸序列依次可如序列表的序列295至序列373所示。所述引物组Ⅶ中14个单链DNA分子的核苷酸序列依次可如序列表的序列374至序列387所示。所述引物组Ⅷ中15个单链DNA分子的核苷酸序列依次可如序列表的序列388至序列402所示。
所述引物组Ⅰ具体可由所述78个单链DNA分子组成。
所述引物组Ⅱ具体可由所述82个单链DNA分子组成。
所述引物组Ⅲ具体可由其所述14个单链DNA分子组成。
所述引物组Ⅳ具体可由其所述15个单链DNA分子组成。
所述引物组Ⅴ具体可由所述75个单链DNA分子组成。
所述引物组Ⅵ具体可由所述79个单链DNA分子组成。
所述引物组Ⅶ具体可由其所述14个单链DNA分子组成。
所述引物组Ⅷ具体可由其所述15个单链DNA分子组成。
上述任一所述试剂盒具体可由上述任一所述接头混合物和所述引物组合组成。
上述任一所述引物组合具体可由所述引物组Ⅰ、所述引物组Ⅱ、所述引物组Ⅲ、所述引物组Ⅳ、所述引物组Ⅴ、所述引物组Ⅵ、所述引物组Ⅶ和所述引物组Ⅷ组成。
上述任一所述试剂盒还可包括用于DNA提取的试剂、用于DNA建库的试剂、用于文库纯化的试剂、用于文库捕获的试剂等用于文库构建的材料。
本发明还保护上述任一所述引物组合。所述引物组合的用途可为检测DNA 样本中肿瘤突变和/或甲基化。
本发明还保护S1)或S2)或S3)：
S1)上述任一所述的引物组合在制备用于检测DNA样本中肿瘤突变和/或甲基化的试剂盒中的应用；
S2)上述任一所述的引物组合在区分肿瘤患者血液样本和非肿瘤患者血液样本中的应用；
S3)上述任一所述试剂盒在区分肿瘤患者血液样本和非肿瘤患者血液样本中的应用。
上述应用中，所述肿瘤可为肝脏恶性肿瘤，即肝细胞癌。
本发明还保护一种检测DNA样本中目标突变和/或甲基化的方法，可包括如下步骤：
(1)按照上述任一所述的方法构建文库；
(2)对步骤(1)得到的文库进行两轮巢式PCR扩增，对产物进行测序，根据测序结果分析DNA样本中目标突变和/或甲基化发生情况；
所述步骤(2)中，采用引物组合甲进行第一轮PCR扩增；
引物组合甲由上游引物甲和下游引物组合甲组成；
所述上游引物甲为文库扩增引物，用于步骤(1)的文库扩增；
所述下游引物组合甲为根据X个目标靶点设计的Y条引物的组合；X和Y均为1以上的自然数，且X≤Y；
以第一轮PCR的产物为模板，采用引物组合乙进行第二轮PCR扩增；
引物组合乙由上游引物乙、下游引物组合乙和index引物组成；
所述上游引物乙为文库扩增引物且3’末端与所述上游引物甲部分相同，用于第一轮PCR的产物的扩增；
所述index引物自5’端包括用于测序的区段A、用于区分样本的index序列和用于测序的区段B；
所述下游引物组合乙中的引物具有所述区段B且与下游引物组合甲中检测相同目标靶点的引物形成巢式关系。
所述上游引物乙的核苷酸序列可如序列表的序列217所示。
所述index引物自5’端具体可由所述区段A、所述index序列和所述区段B组成。
所述区段A的核苷酸序列可如序列表的序列218所示。
所述区段B的核苷酸序列可如序列表的序列219所示。
所述上游引物甲的部分序列与“每个接头的上游引物甲的测序接头甲”的序列完全相同。
所述上游引物乙用于补全文库分子的接头序列，使得扩增产物可以直接进行测序。所述上游引物乙和所述上游引物甲(第一轮PCR扩增使用的引物)的部分核苷酸序列完全相同。
所述上游引物甲的核苷酸序列具体可如序列表的序列27所示。
所述上游引物乙的核苷酸序列具体可如序列表的序列188所示。
当所述目标突变为肝细胞癌突变时，所述下游引物组合甲由上述任一所述引物组Ⅰ和引物组Ⅱ组成。所述下游引物组合乙由上述任一所述引物组Ⅴ和引物组Ⅵ组成。使用引物组Ⅰ和引物组Ⅱ分别对模板进行第一轮PCR扩增。使用引物组Ⅰ进行扩增的产物作为第二轮扩增的模板采用引物组Ⅴ进行扩增。使用引物组Ⅱ进行扩增的产物作为第二轮扩增的模板采用引物组Ⅵ进行扩增。最后将扩增产物等体积混合。
当所述目标甲基化为肝细胞癌甲基化时，所述下游引物组合甲由上述任一所述引物组Ⅲ和引物组Ⅳ组成。所述下游引物组合乙由上述任一所述引物组Ⅶ和引物组Ⅷ组成。
使用引物组Ⅲ和引物组Ⅳ分别对模板进行第一轮PCR扩增。使用引物组Ⅲ进行扩增的产物作为第二轮扩增的模板采用引物组Ⅶ进行扩增。使用引物组Ⅳ进行扩增的产物作为第二轮扩增的模板采用引物组Ⅷ进行扩增。最后将扩增产物等体积混合。
上述方法中，所述DNA样本中目标突变的分析方法可为：将测序数据满足标准甲的DNA分子回溯到一个分子簇；将满足标准乙的分子簇标记为一对duplex分子簇；对某一突变来说，如果满足下述(a1)或(a2)，则该突变为来自原始DNA样本的真实突变：(a1)至少有一对duplex分子簇支持(该条件只支持捕获测序的数据，对于race的数据不适用)；(a2)至少有4个分子簇支持；标准甲即同时满足①、②和③；①DNA插入片段长度相同且除突变位点外序列一致；②随机标签序列相同；③锚定序列相同；标准乙即同时满足④和⑤；④DNA插入片段长度相同且除突变位点外序列一致；⑤分子簇两端的锚定序列相同但位置相反。
上述方法中，所述DNA样本中甲基化的分析方法可为：将测序数据满足标准丙的DNA分子标记为一个簇，分别计算片段末端为关注酶切位点的簇的数量，记录为未甲基化的片段；计算扩增片段达到或超过第一个酶切位点的全部簇的数量，记录为片段总数；根据两种片段数量计算对应区域的平均甲基化水平；区域的甲基化水平＝(1-未甲基化片段数/片段总数)×100％；标准丙即同时满足⑥、⑦和⑧；⑥随机标签序列相同；⑦锚定序列相同；⑧DNA插入片段长度相同且除突变位点外序列一致。
以上所述DNA插入片段具体指除了接头以外的扩增出来的DNA片段。
本发明还保护一种检测DNA样本中多种目标突变和/或甲基化的方法，可包括如下步骤：
(1)按照上述任一所述的方法构建文库；
(2)对步骤(1)的文库进行靶区域富集并进行测序，根据测序结果分析DNA样本中目标突变和/或甲基化的发生情况。
上述方法中，所述DNA样本中目标突变的分析方法可为：将测序数据满足标准甲的DNA分子回溯到一个分子簇；将满足标准乙的分子簇标记为一对duplex分子簇；对某一突变来说，如果满足下述(a1)或(a2)，则该突变为来自原始DNA样本的真实突变：(a1)至少有一对duplex分子簇支持；(a2)至少有4个分子簇支持；标准甲即同时满足①、②和③；①DNA插入片段长度相同且除突变位点外序列一致；②随机标签序列相同；③锚定序列相同；标准乙即同时满足④和⑤；④DNA插入片段长度相同且除突变位点外序列一致；⑤分子簇两端的锚定序列相同但位置相反。
上述方法中，所述DNA样本中甲基化的分析方法可为：将测序数据满足标准丙的DNA分子标记为一个簇，分别计算片段末端为关注酶切位点的簇的数量，记录为未甲基化的片段；计算扩增片段达到或超过第一个酶切位点的全部簇的数量，记录为片段总数；根据两种片段数量计算对应区域的平均甲基化水平；区域的甲基化水平＝(1-未甲基化片段数/片段总数)×100％；标准丙即同时满足⑥、⑦和⑧；⑥随机标签序列相同；⑦锚定序列相同；⑧DNA插入片段长度相同且除突变位点外序列一致。
所述靶区域富集可采用现有商购的靶向捕获试剂盒(例如Agilent sureselect XT靶向捕获试剂盒，Agilent5190-8646)进行，将其中最后一步PCR扩增的引物对更换为由引物甲和引物乙组成的引物对。所述引物甲的核苷酸序列可如序列表的序列403所示。所述引物乙可包括区段甲、index序列和区段乙。所述引物乙具体可由所述区段甲、所述index序列和所述区段乙组成。所述区段甲的核苷酸序列可如序列表的序列404所示。所述区段乙的核苷酸序列可如序列表的序列405所示。
上述任一所述方法中，所述目标突变和/或甲基化可为肿瘤突变和/或甲基化。所述肿瘤可为肝脏恶性肿瘤，即肝细胞癌。
上文中，通常多个不同样本的文库会混合在一起测序，所述index序列用于标记不同的样本。测序完成后，根据不同的index序列对总的测序数据进行拆分。Index的设计原则与之前描述的锚定序列的设计原则基本相似。
上文中，DNA样本用甲基化敏感限制性内切酶酶切后，形成DNA片段(此时的DNA片段两端均形成粘末端，末端的单链部分的核苷酸序列即为断点序列)；DNA片段进行末端修复后与接头连接(5’末端和3’末端各连接一个接头，可能是相同接头也可能是相反接头)，对于此时的DNA分子来说，两个接头之间的DNA片段即为DNA插入片段。
本发明提供了一种可以在一份样本中同时检测ctDNA中的肿瘤特异基因的变异(包括点突变、插入缺失突变、HBV整合等多种突变形式)和/或甲基化的方法，不仅样本量需求低，而且该方法制备的MC文库可以支持10-20次的后续检测，每次检测的结果都可以代表全部原始ctDNA标本的突变状况及酶切位点覆盖区域的甲基化修饰情况，且不会造成灵敏度和特异性的降低。使用该方法 构建的文库可以同时用于PCR的热点检测和捕获法测序，加入的DNA barcode可以有效滤除假阳性结果，实现基于duplex高特异性测序。同时，文库的构建方法不仅适用于cfDNA样本，也适用于基因组DNA或cDNA样本。本发明对肿瘤早期筛查、病情追踪、疗效评估、预后预测等具有重要的临床意义，有重大的应用价值。
附图说明
图1为adapter和引物架构示意图。
图2为RaceSeq靶区富集和文库构建示意图。
图3为MC文库进行捕获和进行duplex测序的示意图。
图4为Padlock方法和突变/甲基化共检法(即本发明提供的方法)对AK055957基因的甲基化水平检测结果。
图5为单独检测突变法和突变/甲基化共检法检测突变和突变频率的结果。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。
以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
下述实施例中的TE缓冲液为ThermoFisher公司的产品，产品目录号为12090015。
下述实施例中，肝细胞癌患者对本发明进行的内容均知情同意。
实施例1、MC文库的构建
一、甲基化敏感限制性内切酶酶切
取5-40ng cfDNA，按照表1所示配置反应体系，然后按照表2的程序在PCR仪进行酶切处理，得到酶切产物(4℃保存)。
Restriction Enzyme和Restriction Enzyme 10×Buffer均为ThermoFisher公司的产品。Restriction Enzyme和Restriction Enzyme 10×Buffer可以根据不同的待测靶标区域进行选择，选择标准为在待测区域内含有至少1个该甲基化敏感限制性内切酶的酶切位点。
表1.反应体系
成分 体积
cfDNA 16.8μl
Restriction Enzyme 10×Buffer 2μl
Acetylated BSA(浓度为10μg/μl) 0.2μl
Restriction Enzyme(浓度为10U/μl) 1μl
总体积 20μl
表2.反应程序
温度 时间
37℃ 2h
二、酶切产物的纯化
取步骤一得到的酶切产物，采用Apostle MiniMax TM高效游离DNA富集分离试剂盒(标准版)(Apostle公司的产品，产品目录号为A17622-50)进行纯化、富集，得到纯化产物。
三、纯化产物的平末端修复和加A处理
取步骤二得到的纯化产物，按照表3所示配置反应体系，然后按照表4的反应程序在PCR仪进行末端修复及3’末端加A处理，得到反应产物(4℃保存)。
表3.反应体系
成分 体积
纯化产物 50μl
End Repair&A-Tailing Buffer(KAPA KK8505) 7μl
End Repair&A-Tailing Enzyme Mix(KAPA KK8505) 3μl
总体积 60μl
表4.反应程序
温度 时间
20℃ 30min
65℃ 30min
四、反应产物与adapter连接
按照表5配置反应体系，20℃反应15min，得到连接产物(4℃保存)。
表5.反应体系
成分 体积
步骤三得到的反应产物 60μl
Adapter Mix(50μM) 1.5μl
DNase/RNase-Free Water 8.5μl
Ligation Buffer(KAPA KK8505) 30μl
DNA Ligase(KAPA KK8505) 10μl
总体积 110μl
Adapter序列信息见表6。
分别将表6中的单链DNA分子用TE缓冲液溶解并稀释至浓度为100μM。将同一组中的两条单链DNA分子等体积混合(各50μl)，然后进行退火(退火程序：95℃，15min；25℃，2h)，得到12组DNA溶液，将12组DNA溶液等体积混合，得到Adapter Mix。
表6.Adapter序列信息
Figure PCTCN2020120560-appb-000001
Figure PCTCN2020120560-appb-000002
表6中，8个N表示8bp的随机标签。实际应用中，随机标签长度可为8-14bp。
下划线表示12bp的锚定序列，每一组的上游序列(名称中含有“F”的为上游序列)和下游序列(名称中含有“R”的为下游序列)中，下划线部分反向互补，通过退火可使上游序列和下游序列结合在一起形成接头。同时，锚定序列可作为序列固定的内置标签，用于标记原始模板分子。实际应用中，锚定序列长度可为12-20bp，连续重复碱基不超过3个，且不能与引物其它部分相互作用(如形成发卡结构、二聚体等)，12组每一个位置碱基平衡(即A、T、C和G均匀分布)，错配碱基数≥3(即各个锚定序列之间的碱基至少有3个不同；不同可为位置不同或顺序不同)。
上游序列中末端加粗的T与原始分子末端加的“A”互补，进行TA连接。
上游序列中，自5’端第1至21位(来自Illumina公司的Truseq测序试剂盒)为测序引物结合序列，其中，自5’端第1至19位为文库扩增引物部分。
下游序列中，非下划线部分(来自Illumina公司的nextera测序试剂盒)为测序引物结合序列，其中，自3’端第1至22位为文库扩增引物的部分。
表6中共包含12组接头，可以形成12×12＝144种标记组合，结合分子本身的序列信息，足以区分原始样品中的所有分子，实际应用中也可适当增加(合成成本增高)或减少(区分效果略弱)组数。
连接产物结构如图1所示。其中，a为接头部分，b和f分别为文库扩增引物，c为8bp随机标签(表6中的8个N表示)，d为12bp锚定序列(表6中 的下划线表示)，e为插入片段(cfDNA)。
五、连接产物纯化
向步骤四得到的连接产物中加入110-220μl(即1-2倍体积)的AMPure XP磁珠(贝克曼A63880)，涡旋混匀，室温放置10min，磁力架吸附5min；待溶液澄清后弃上清，然后加入200μl 80％(体积百分含量)乙醇水溶液清洗2次，弃上清；待乙醇晾干后，加入30μl DNase/RNase-Free Water，涡旋混匀，室温放置10min，磁力架吸附5min，吸取上清溶液至PCR管内，作为PCR模板。
六、文库扩增及纯化
1、取步骤五得到的PCR模板，按照表7配置反应体系，按照表8进行PCR扩增，得到PCR扩增产物(4℃保存)。
表7.反应体系
成分 体积
HIFI(KAPA KK8505) 35μl
MC_F(33μM) 2.5μl
MC_R(33μM) 2.5μl
PCR模板 30μl
总体积 70μl
表7中，引物信息如下：
MC_F(序列25)：5’-GACACGACGCTCTTCCGAT-3’；
MC_R(序列26)：5’-GTGGGCTCGGAGATGTGTATAA-3’。
表8.反应程序
Figure PCTCN2020120560-appb-000003
2、向步骤1得到的PCR扩增产物中加入70-140μl(即1-2倍体积)的AMPure XP磁珠，涡旋混匀，室温放置10min，磁力架吸附5min；待溶液澄清后弃上清，然后加入200μl 80％(体积百分含量)乙醇水溶液清洗2次，弃上清；待乙醇晾干后，加入100μl DNase/RNase-Free Water，涡旋混匀，室温放置10min，磁力架吸附5min，吸取上清溶液，得到产物(-20℃储存)。产物即为可长期保存、反复使用的MC文库。
经检测，MC文库可以支持10-20次的后续检测，每次检测的结果都可以代表全部原始样本的突变状况及酶切位点覆盖区域的甲基化修饰情况，不会造成灵敏度和特异性的降低。同时，文库的构建方法不仅适用于cfDNA样本，也适用于基因组DNA或cDNA样本。
实施例2、RaceSeq富集靶区并构建测序文库
如图2所示，使用针对我国肝细胞癌高频突变基因(TP53、CTNNB1、AXIN1、TERT)的相关区域、HBV整合热点区域和肝细胞癌特异高甲基化区域(EMX1、LRRC4、BDH1等)设计的引物，和固定引物配合，对MC文库进行两轮PCR扩增，扩增产物即为测序文库。
图2中，a为第一轮文库扩增上游引物，b为第二轮文库扩增上游引物，c为第一轮文库扩增下游引物库，用于特异目标序列富集，d为第二轮文库扩增下游引物库，用于特异目标序列富集，e为index primer，用于添加index序列。
1、取300ng实施例1制备的MC文库，分为两份，配置表9的反应体系(一份加入GSP1A mix，另一份加入GSP1B mix)，按照表11的反应程序进行第一轮PCR扩增，得到第一轮扩增产物(共得到两份第一轮扩增产物，一份为GSP1A mix的扩增产物，一份为GSP1B mix的扩增产物)。
表9.反应体系
成分 体积
Hifi(KAPA KK8505) 15μl
上游引物1355 3μl
GSP1A mix/GSP1B mix 2μl
MC文库 10μl
总体积 30μl
表9中，引物信息如下：
上游引物1355(序列27)：5’-TCTTTCCCTACACGACGCTCTTCCGAT-3’。
GSP1A mix：将表10中属于引物池GSP1A中的各条引物用TE缓冲液溶解并稀释至浓度为100μM，然后等体积混合，用TE缓冲液稀释至0.3μM。引物池GSP1A中的引物用于扩增模板的正链。
GSP1B mix：将表10中属于引物池GSP1B中的各条引物用TE缓冲液溶解并稀释至浓度为100μM，然后等体积混合，用TE缓冲液稀释至0.3μM。引物池GSP1B中的引物用于扩增模板的负链。
引物池GSP1A和引物池GSP1B中，相同编号的引物(即引物编号的后四位相同)从正负两个方向检测同一突变位点，同时使用可最大限度的富集原始分子信息。
表10.引物信息
Figure PCTCN2020120560-appb-000004
Figure PCTCN2020120560-appb-000005
Figure PCTCN2020120560-appb-000006
Figure PCTCN2020120560-appb-000007
Figure PCTCN2020120560-appb-000008
Figure PCTCN2020120560-appb-000009
表11.反应程序
Figure PCTCN2020120560-appb-000010
2、分别将步骤1得到的两份第一轮扩增产物用30-60μl(即1-2倍体积)的AMPure XP磁珠进行纯化，然后用25μlDNase/RNase-Free Water洗脱，得到第一轮纯化产物。
3、分别以步骤2得到的第一轮纯化产物为模板，配置表12的反应体系(采用GSP1A mix扩增产物作为模板时，采用GSP2A mix扩增；采用GSP1Bmix扩增产物作为模板时，采用GSP2B mix扩增)，按照表14的反应程序进行第二轮PCR扩增，得到第二轮扩增产物(4℃保存)。
表12.反应体系
成分 体积
KapaHifi 15μl
上游引物3355 2μl
GSP2Amix/GSP2Bmix 1μl
Index引物(10μM) 2μl
模板(GSP1A mix/GSP1Bmix) 10μl
总体积 30μl
表12中，引物信息如下：
上游引物3355(序列217)：
5’-AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCT-3’；下划线部分为第一轮的上游引物1355相同的部分，3355及1355均为Illumina测序平台测序的固定序列(也可更换为可在其他测序平台测序的序列)。
GSP2A mix：将表13中属于引物池GSP2A中的各条引物用TE缓冲液溶解并稀释至浓度为100μM，然后等体积混合，用TE缓冲液稀释至0.3μM。引物池GSP2A中的引物用于扩增模板的正链。
GSP2B mix：将表13中属于引物池GSP2B中的各条引物用TE缓冲液溶解并稀释至浓度为100μM，然后等体积混合，用TE缓冲液稀释至0.3μM。引物池GSP2B中的引物用于扩增模板的负链。
表13中，自5’端第1至15位为与Index引物结合的部分。
GSP2A mix与GSP1A mix中引物编号相同的引物(即引物编号的后四位相同)为针对同一突变位点设计，两条引物形成巢式关系。
GSP2B mix与GSP2A mix中引物编号相同的引物(即引物编号的后四位相同)为针对同一突变位点设计，两条引物形成巢式关系。
Index引物：5’-CAAGCAGAAGACGGCATACGAGAT(序列218)********GTGACTGGAGTTC CTTGGCACCCGAGAA-3’(序列219)；下划线部分为与GSP2mix结合的部分。********为index序列位置，index的长度为6-8bp，作用是区分样本间的序列，方便多个样本混合测序。除index序列外，其余都是来自Illumina的small RNA测序试剂盒的固定序列。
表13.引物信息
Figure PCTCN2020120560-appb-000011
Figure PCTCN2020120560-appb-000012
Figure PCTCN2020120560-appb-000013
Figure PCTCN2020120560-appb-000014
Figure PCTCN2020120560-appb-000015
Figure PCTCN2020120560-appb-000016
注：NA表示无引物。
表14.反应程序
Figure PCTCN2020120560-appb-000017
4、步骤3得到的采用GSP2A mix进行第二轮扩增的产物和采用GSP1B mix进行第二轮扩增的产物等体积混合，利用AMPure XP磁珠按1：(1-2)的比例进行纯化，然后用50μl DNase/RNase-Free Water洗脱，得到第二轮纯化产物，即为可以在Illumina Hiseq X平台测序的测序文库。
MC文库上的DNA随机标签与cfDNA序列一起被添加到测序文库的Read1序列下游。在测序中，依次获得DNA随机标签序列、锚定序列、cfDNA序列(图1中的c、d、e序列)。
肝细胞癌特异基因变异的分析方法为：将测序数据满足标准甲的DNA分子回溯到一个分子簇；将满足标准乙的分子簇标记为一对duplex分子簇；对某一突变来说，如果满足下述(a1)或(a2)，则该突变为来自原始DNA样本的真实突变：(a1)至少有一对duplex分子簇支持；(a2)至少有4个分子簇支持；标准甲即同时满足①、②和③；①DNA插入片段长度相同且除突变位点外序列一 致；②随机标签序列相同；③锚定序列相同；标准乙即同时满足④和⑤；④DNA插入片段长度相同且除突变位点外序列一致；⑤分子簇两端的锚定序列相同但位置相反。
肝细胞癌特异甲基化修饰程度的分析方法为：将测序数据满足标准丙的DNA分子标记为一个簇，分别计算片段末端为关注酶切位点的簇的数量，记录为未甲基化的片段；计算扩增片段达到或超过第一个酶切位点的全部簇的数量，记录为片段总数；根据两种片段数量计算对应区域的平均甲基化水平；区域的甲基化水平＝(1-未甲基化片段数/片段总数)×100％；标准丙即同时满足⑥、⑦和⑧；⑥随机标签序列相同；⑦锚定序列相同；⑧DNA插入片段长度相同且除突变位点外序列一致。
实施例3、MC文库的捕获及测序
如图3所示，靶区域富集可基于现有商业化的靶向捕获试剂盒进行优化设计而捕获。例如：基于甲基化区域的捕获可参考Roche SeqCap Epi CpGiant Enrichment Kit(Roche 07138881001)或Illumina Infinium Methylation EPIC BeadChipWG-317-1001)，甲基化区域靶向捕获的设计需要根据酶切位点覆盖程度进行筛选，并调整探针中基于重亚硫酸盐处理所转换的碱基。基于基因变异区域的捕获可参考Agilent sureselect XT靶向捕获试剂盒(Agilent5190-8646)，仅将最后一步PCR扩增的引物更换为如下引物：
上游引物为：5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCT ACACGACGCTCTTCCGATCT-3’(序列403)(图3中的“a”)，下划线部分与引物MC_F部分相同)，作用为扩增文库，其余部分为illumina测序平台测序所需的固定序列。
下游引物为：5’-CAAGCAGAAGACGGCATACGAGAT(序列404)********GTCTC GTGGGCTCGGAGATGTGTATAA-3’(序列405)(图3中的“b”)，下划线部分为与引物MC_R相同，作用为扩增文库。********为index序列位置，index长度为6-8bp，作用是区分样本间的序列，方便多个样本混合测序。其余部分为illumina测序平台测序所需的固定序列。
捕获后的文库与MC文库具有相同的DNA随机标签序列、锚定序列和cfDNA序列，依次位于Read1下游。
将测序数据满足标准甲的DNA分子回溯到一个分子簇；标准甲即同时满足①、②和③；①DNA插入片段长度相同且除突变位点外序列一致；②随机标签序列相同；③锚定序列相同。将满足标准乙的分子簇标记为一对duplex分子簇；标准乙即同时满足④和⑤；④DNA插入片段长度相同且除突变位点外序列一致；⑤分子簇两端的锚定序列相同但位置相反。对某一突变来说，如果满足下述(a1)或(a2)，则该突变为来自原始DNA样本的真实突变：(a1)至少有一对duplex分子簇支持；(a2)至少有4个分子簇支持。在一对duplex分子簇共同支持的突变可靠性更高，可减少90％的假阳性突变。
将测序数据满足标准丙的DNA分子标记为一个簇，分别计算片段末端为关注酶切位点的簇的数量，记录为未甲基化的片段；计算扩增片段达到或超过第一个酶切位点的全部簇的数量，记录为片段总数；根据两种片段数量计算对应区域的平均甲基化水平；区域的甲基化水平＝(1-未甲基化片段数/片段总数)×100％；标准丙即同时满足⑥、⑦和⑧；⑥随机标签序列相同；⑦锚定序列相同；⑧DNA插入片段长度相同且除突变位点外序列一致。
实施例4、检测方法的对比
一、检测方法的对比一
1、收集21例肝细胞癌患者的cfDNA标本。
2、完成步骤1后，取各个cfDNA标本，按照实施例1中的方法构建MC文库，然后按照实施例2中的方法进行RaceSeq靶区富集，测序，得到AK055957基因的甲基化水平。
3、完成步骤1后，取各个cfDNA标本，采用Padlock方法(Xu R H,Wei W,Krawczyk M,et al.Circulating tumour DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma[J].Nature Materials,2017,16(11):1155.)检测AK055957基因的甲基化水平。Padlock是一种甲基化靶向测序技术，Padlock探针构象与挂锁类似，可应用于高通量甲基化靶向测序，属于高效的亚硫酸氢盐转化后建库方法，被称为“BSPP”。cfDNA经亚硫酸氢盐转化后，与亚硫酸盐锁式探针(BSPP)的捕获臂互补配对时可以扩增并连接成环形，用核酸外切酶可以筛选出连接成环状的锁式探针，对扩增产物进行测序得到相应的DNA甲基化信息。
检测结果见图4。结果表明，Padlock方法和突变/甲基化共检法(即本发明提供的方法)对AK055957基因(属于肝细胞癌特异基因)的甲基化水平检测结果基本一致。
二、检测方法的对比二
1、采用突变/甲基化共检法检测突变和突变频率
(1)收集某肝细胞癌患者的cfDNA。
(2)完成步骤(1)后，取5-40ngcfDNA，按照表1所示配置反应体系，然后在PCR仪进行酶切处理，得到酶切产物(4℃保存)。其中酶切处理的时间为0h、0.2h、0.4h、0.6h、0.8h或1h。
(3)完成步骤(2)后，取所述酶切产物，按照实施例1中二至六的方法构建MC文库，然后按照实施例2中的方法进行RaceSeq靶区富集，测序。在数据分析时，将随机标签序列相同、DNA插入片段长度相同且除突变位点外序列一致的DNA分子测序数据回溯到一个分子簇，如果簇内分子数>5个并且簇内分子突变一致率>80％并且簇个数≥5，则该突变为来自原始DNA样本的真实突变。含该分子突变的簇的比例即为突变频率。
2、采用单独检测突变法检测突变和突变频率
(1)收集某肝细胞癌患者的cfDNA。
(2)完成步骤(1)后，取5-40ng cfDNA，按照表3所示配置反应体系，然后按照表4的反应程序在PCR仪进行末端修复及3’末端加A处理，得到反应产物(4℃保存)。
(3)完成步骤(2)后，取所述反应产物，按照实施例1中四至六的方法构建MC文库，然后按照实施例2中的方法进行RaceSeq靶区富集，测序。在数据分析时，将随机标签序列相同、DNA插入片段长度相同且除突变位点外序列一致的DNA分子测序数据回溯到一个分子簇，如果簇内分子数>5个并且簇内分子突变一致率>80％并且簇个数≥5，则该突变为来自原始DNA样本的真实突变。含该分子突变的簇的比例即为突变频率。
3、将各突变位点根据突变/甲基化共检法得到的突变频率作为横坐标，单独检测突变法得到的突变频率作为纵坐标，绘制散点图，添加线性拟合曲线及相关系数R 2。
结果见图5。结果表明，突变/甲基化共检法和单独检测突变法对于突变和突变频率的检测结果基本一致，即甲基化检测并未影响对突变的检出。
实施例5、准确性实验
突变标准品为Horizon Discovery公司的产品，产品目录号为HD701。
一、准确性实验一
1、取突变标准品，按照实施例1中一至六的方法构建MC文库，然后按照实施例2中的方法(仅将步骤3中的GSP2A mix替换为GSP2A mix-1，GSP2B mix替换为GSP2B mix-1)进行RaceSeq靶区富集，测序。
GSP2A mix-1：将表15中属于引物池GSP2A中的各条引物用TE缓冲液溶解并稀释至浓度为100μM，然后等体积混合，用TE缓冲液稀释至0.3μM。引物池GSP2A中的引物用于扩增模板的正链。
GSP2B mix-1：将表15中属于引物池GSP2B中的各条引物用TE缓冲液溶解并稀释至浓度为100μM，然后等体积混合，用TE缓冲液稀释至0.3μM。引物池GSP2B中的引物用于扩增模板的负链。
表15.引物序列
基因名称 染色体 突变位点 引物池 引物编号 引物序列(5’-3’)
PIK3CA 3 178916875 GSP2A HA2094 cagaaagggaagaattttttgatgaaaca
PIK3CA 3 178921551 GSP2A HA2095 ctcagaataaaaattctttgtgcaacctac
PIK3CA 3 178936082 GSP2A HA2096 gctcaaagcaatttctacacgagatc
PIK3CA 3 178952072 GSP2A HA2097 gcaagaggctttggagtatttcatg
KRAS 12 25398285 GSP2A HA2115 tgactgaatataaacttgtggtagttgg
KRAS 12 25380277 GSP2A HA2116 cctgtctcttggatattctcgacac
KRAS 12 25378562 GSP2A HA2117 gcaagaagttatggaattccttttattgaa
EGFR 7 55241707 GSP2A HA2121 ttgaggatcttgaaggaaactgaatt
EGFR 7 55242463 GSP2A HA2122 tgagaaagttaaaattcccgtcgcta
EGFR 7 55249004 GSP2A HA2123 ctccaggaagcctacgtgatg
EGFR 7 55249071 GSP2A HA2124 acctccaccgtgcagctc
EGFR 7 55259514 GSP2A HA2125 ccgcagcatgtcaagatcacag
PIK3CA 3 178916875 GSP2B HB2094 ggttgaaaaagccgaaggtcac
PIK3CA 3 178921551 GSP2B HB2095 catttgactttaccttatcaatgtctcgaa
PIK3CA 3 178936082 GSP2B HB2096 acttacctgtgactccatagaaaatctt
PIK3CA 3 178952072 GSP2B HB2097 caatccatttttgttgtccagcc
KRAS 12 25398285 GSP2B HB2115 tagctgtatcgtcaaggcactc
KRAS 12 25380277 GSP2B HB2116 ggtccctcattgcactgtact
KRAS 12 25378562 GSP2B HB2117 tgtatttatttcagtgttacttacctgtcttg
EGFR 7 55241707 GSP2B HB2121 accttatacaccgtgccgaa
EGFR 7 55242463 GSP2B HB2122 actcacatcgaggatttccttgtt
EGFR 7 55249004 GSP2B HB2123 cggtggaggtgaggcagat
EGFR 7 55249071 GSP2B HB2124 gtccaggaggcagccgaa
EGFR 7 55259514 GSP2B HB2125 gtattctttctcttccgcaccca
2、根据测序结果，得到突变位点的突变频率。
检测结果见表16。结果表明，采用突变/甲基化共检法检测突变标准品，得到的突变位点的突变频率与理论值基本接近。由此可见，突变/甲基化共检法对肝细胞癌特异基因(如CTNNB1基因、TP53基因、AXIN1基因)的变异检测具有较高的准确性。
表16.准确性实验
Figure PCTCN2020120560-appb-000018
注：geneID代表基因在Ensemble数据库中编号，Ref为正常类型，Alt为基因变异后类型，INS代表插入，DEL代表缺失，SNP代表单碱基突变。
二、准确性实验二
人甲基化和非甲基化标准品为Zymo Research公司的产品，产品目录号为D5014。
1、将人甲基化和非甲基化标准品中的甲基化标准品和非甲基化标准品按照不同比例进行混合，得到待测样本。待测样本中，甲基化标准品的比例为0％、 20％或100％，即肿瘤特异基因(BDH1基因、EMX1基因、LRRC4基因、CLEC11A基因、HOXA1基因、AK055957基因、COTL1基因、ACP1基因或DAB2IP基因)甲基化的比例为0％、20％或100％。
2、取待测样本，按照实施例1中的方法构建MC文库，然后按照实施例2中的方法进行RaceSeq靶区富集，测序，得到甲基化位点的检测值。
检测结果见表17和表18(样本类型的后四位为肿瘤特异基因的名称)。采用突变/甲基化共检法检测甲基化标准品，检测值与理论值基本接近。由此可见，突变/甲基化共检法对肿瘤特异基因(如BDH1基因、EMX1基因、LRRC4基因、CLEC11A基因、HOXA1基因、AK055957基因、COTL1基因、ACP1基因、DAB2IP基因)的甲基化水平检测具有较高的准确性。
表17.甲基化标准品准确度检测结果(正链)
样本类型 0％甲基化标准品 20％甲基化标准品 100％甲基化标准品
CA2001_BDH1 2％ 18％ 97％
CA2002_EMX1 3％ 19％ 96％
CA2003_LRRC4 2％ 9％ 100％
CA2004_LRRC4 3％ 32％ 97％
CA2006_CLEC11A 2％ 20％ 97％
CA2007_CLEC11A 2％ 25％ 99％
CA2008_HOXA1 3％ 20％ 99％
CA2009_HOXA1 3％ 23％ 99％
CA2010_EMX1 3％ 32％ 99％
CA2011_AK055957 3％ 23％ 99％
CA2012_COTL1 3％ 18％ 98％
CA2013_ACP1 4％ 27％ 98％
CA2014_DAB2IP 2％ 21％ 98％
表18.甲基化标准品准确度检测结果(负链)
样本类型 0％甲基化标准品 20％甲基化标准品 100％甲基化标准品
CB2001_BDH1 3％ 21％ 96％
CB2002_LRRC4 3％ 17％ 98％
CB2004_LRRC4 2％ 9％ 96％
CB2005_DAB2IP 2％ 3％ 99％
CB2007_CLEC11A 4％ 50％ 94％
CB2008_CLEC11A 3％ 18％ 97％
CB2009_HOXA1 2％ 20％ 98％
CB2011_EMX1 3％ 23％ 99％
CB2012_AK055957 4％ 19％ 100％
CB2013_RASSF2 7％ 60％ 94％
CB2015_DAB2IP 3％ 23％ 99％
实施例6、突变/甲基化共检法在肝细胞癌患者cfDNA中的应用
1、收集1例正常人、1例肝硬化患者和3例肝细胞癌患者的血液样本，提取cfDNA。
2、取5-40ng cfDNA，按照实施例1进行MC文库构建，按照实施例2中的方法进行RaceSeq靶区富集，测序。
3、甲基化检测结果见表19和表20。
结果显示肝细胞癌特异高甲基化的基因，在所检测的肝细胞癌样本中的甲基化水平高于非肝细胞癌样本中的甲基化水平。突变/甲基化共检法可以应用于肝细胞癌cfDNA样本的检测。
表19.cfDNA样本靶标区域甲基化水平检测结果(正链)
样本类型 正常 肝硬化 肝细胞癌1 肝细胞癌2 肝细胞癌3
CA2001_BDH1 3％ 3％ 28％ 25％ 47％
CA2002_EMX1 4％ 6％ 11％ 26％ 4％
CA2003_LRRC4 3％ 5％ 16％ 28％ 28％
CA2004_LRRC4 3％ 6％ 29％ 46％ 48％
CA2006_CLEC11A 3％ 4％ 11％ 20％ 2％
CA2007_CLEC11A 3％ 5％ 22％ 25％ 10％
CA2008_HOXA1 4％ 4％ 24％ 33％ 5％
CA2009_HOXA1 8％ 7％ 10％ 11％ 11％
CA2010_EMX1 7％ 9％ 21％ 47％ 8％
CA2011_AK055957 5％ 9％ 40％ 43％ 45％
CA2012_COTL1 5％ 9％ 17％ 19％ 5％
CA2013_ACP1 1％ 3％ 5％ 5％ 14％
CA2014_DAB2IP 5％ 7％ 19％ 27％ 50％
表20.cfDNA样本靶标区域甲基化水平检测结果(负链)
样本类型 正常 肝硬化 肝细胞癌1 肝细胞癌2 肝细胞癌3
CB2001_BDH1 5％ 5％ 24％ 23％ 56％
CB2002_LRRC4 4％ 13％ 40％ 47％ 50％
CB2004_LRRC4 1％ 4％ 11％ 17％ 28％
CB2005_DAB2IP 4％ 5％ 10％ 16％ 27％
CB2007_CLEC11A 11％ 8％ 17％ 38％ 6％
CB2008_CLEC11A 2％ 5％ 22％ 23％ 7％
CB2009_HOXA1 4％ 2％ 10％ 21％ 3％
CB2011_EMX1 12％ 11％ 20％ 39％ 7％
CB2012_AK055957 3％ 9％ 39％ 38％ 43％
CB2013_RASSF2 5％ 1％ 4％ 18％ 4％
CB2015_DAB2IP 9％ 6％ 18％ 31％ 57％
工业应用
本发明公开了一种可以在一份样本中同时检测ctDNA中的肿瘤特异基因的变异(包括点突变、插入缺失突变、HBV整合等多种突变形式)和/或甲基化的方法，不仅样本量需求低，而且该方法制备的MC文库可以支持10-20次的后续检测，每次检测的结果都可以代表全部原始ctDNA标本的突变状况及酶切位点覆盖区域的甲基化修饰情况，且不会造成灵敏度和特异性的降低。同时，文库的构建方法不仅适用于cfDNA样本，也适用于基因组DNA或cDNA样本。本发明对肿瘤早期筛查、病情追踪、疗效评估、预后预测等具有重要的临床意义，有重大的应用价值。

Claims (15)

  1. 一种测序文库构建方法，依次包括如下步骤：
    (1)取DNA样本，用甲基化敏感限制性内切酶酶切；
    (2)将步骤(1)酶切后的DNA样本依次进行末端修复和3’端加A处理；
    (3)将步骤(2)处理后的DNA样本与接头混合物中的接头连接，经过PCR扩增后得到文库；
    所述接头混合物由n个接头组成；
    每个接头均由一条上游引物甲和一条下游引物甲形成部分双链结构得到；上游引物甲中具有测序接头甲、随机标签、锚定序列甲和位于末端的碱基T；下游引物甲中具有锚定序列乙和测序接头乙；所述部分双链结构由锚定序列甲和锚定序列乙反向互补形成；
    所述测序接头甲和测序接头乙为根据不同测序平台选择对应的测序接头；
    所述随机标签为8-14bp的随机碱基；
    所述锚定序列甲长度为12-20bp，连续重复碱基≤3个；
    n个接头采用n个不同的锚定序列甲，且每个锚定序列甲中四种碱基平衡，错配碱基数≥3；
    n为≥8的任意自然数。
  2. 如权利要求1所述的构建方法，其特征在于：
    所述上游引物甲自5’端依次包括所述测序接头甲、所述随机标签、所述锚定序列甲和所述碱基T；
    所述下游引物甲自5’端依次包括所述锚定序列乙和所述测序接头乙。
  3. 如权利要求1所述的构建方法，其特征在于：所述错配碱基数≥3为所述接头混合物包含n个锚定序列甲，各个锚定序列甲之间的碱基至少有3个不同；所述不同为位置不同或顺序不同。
  4. 如权利要求1所述的构建方法，其特征在于：所述DNA样本为基因组DNA、cDNA、ct DNA或cf DNA样本。
  5. 权利要求1至4任一所述方法构建得到的DNA文库。
  6. 一种用于构建测序文库的试剂盒，包括权利要求1至4中所述的接头混合物和甲基化敏感限制性内切酶。
  7. 一种用于检测DNA样本中肿瘤突变和/或甲基化的试剂盒，包括权利要求1至4中所述接头混合物和引物组合；所述引物组合包括引物组Ⅰ、引物组Ⅱ、引物组Ⅲ、引物组Ⅳ、引物组Ⅴ、引物组Ⅵ、引物组Ⅶ和引物组Ⅷ；
    所述引物组Ⅰ和所述引物组Ⅱ中的各个引物是根据与肿瘤突变相关的区域设计的特异性引物，作用是定位于基因组特定位置，实现目标区域的PCR富集；所述引物组Ⅰ和所述引物组Ⅱ分别用于检测DNA正链和负链的突变位点；
    所述引物组Ⅲ和所述引物组Ⅳ中的各个引物是根据肿瘤特异高甲基化区域设计的特异性引物，作用是定位于基因组特定位置，实现目标区域的PCR富集； 所述引物组Ⅲ和所述引物组Ⅳ分别用于检测DNA正链和负链的甲基化位点；
    所述引物组Ⅴ、所述引物组Ⅵ、所述引物组Ⅶ和所述引物组Ⅷ中的各个引物均包括接头序列和特异序列，特异序列用于目标区域进行进一步富集；
    所述引物组Ⅴ和所述引物组Ⅰ中，针对同一突变位点设计的两个引物为“巢式”关系；
    所述引物组Ⅵ和所述引物组Ⅱ中，针对同一突变位点设计的两个引物为“巢式”关系；
    所述引物组Ⅶ和所述引物组Ⅲ中，针对同一甲基化位点设计的两个引物为“巢式”关系；
    所述引物组Ⅷ和所述引物组Ⅳ中，针对同一甲基化位点设计的两个引物为“巢式”关系。
  8. 如权利要求7所述的试剂盒，其特征在于：所述肿瘤为肝脏恶性肿瘤。
  9. 如权利要求8所述的试剂盒，其特征在于：
    所述引物组Ⅰ包括78个单链DNA分子，78个单链DNA分子的核苷酸序列依次如序列表的序列28至序列105所示；
    所述引物组Ⅱ包括82个单链DNA分子，82个单链DNA分子的核苷酸序列依次如序列表的序列106至序列187所示；
    所述引物组Ⅲ包括14个单链DNA分子，14个单链DNA分子的核苷酸序列依次如序列表的序列188至序列201所示；
    所述引物组Ⅳ包括15个单链DNA分子，15个单链DNA分子的核苷酸序列依次如序列表的序列202至序列216所示；
    所述引物组Ⅴ包括75个单链DNA分子，75个单链DNA分子依次包括如序列表的序列220至序列294自5’末端起第16位至3’末端所示的核苷酸序列；
    所述引物组Ⅵ包括79个单链DNA分子，79个单链DNA分子依次包括如序列表的序列295至序列373自5’末端起第16位至3’末端所示的核苷酸序列；
    所述引物组Ⅶ包括14个单链DNA分子，14个单链DNA分子依次包括如序列表的序列374至序列387自5’末端起第16位至3’末端所示的核苷酸序列；
    所述引物组Ⅷ包括15个单链DNA分子，15个单链DNA分子依次包括如序列表的序列388至序列402自5’末端起第16位至3’末端所示的核苷酸序列。
  10. 权利要求7至9中任一所述的引物组合。
  11. S1)或S2)或S3)：
    S1)权利要求7至9中任一所述的引物组合在制备用于检测DNA样本中肿瘤突变和/或甲基化的试剂盒中的应用；
    S2)权利要求7至9中任一所述的引物组合在区分肿瘤患者血液样本和非肿瘤患者血液样本中的应用；
    S3)权利要求7至9任一所述试剂盒在区分肿瘤患者血液样本和非肿瘤患者血液样本中的应用。
  12. 一种检测DNA样本中目标突变和/或甲基化的方法，包括如下步骤：
    (1)按照权利要求1至4任一所述的方法构建文库；
    (2)对步骤(1)得到的文库进行两轮巢式PCR扩增，对产物进行测序，根据测序结果分析DNA样本中目标突变和/或甲基化发生情况；
    所述步骤(2)中，采用引物组合甲进行第一轮PCR扩增；
    引物组合甲由上游引物甲和下游引物组合甲组成；
    所述上游引物甲为文库扩增引物，用于步骤(1)的文库扩增；
    所述下游引物组合甲为根据X个目标靶点设计的Y条引物的组合；X和Y均为1以上的自然数，且X≤Y；
    以第一轮PCR的产物为模板，采用引物组合乙进行第二轮PCR扩增；
    引物组合乙由上游引物乙、下游引物组合乙和index引物组成；
    所述上游引物乙为文库扩增引物且3’末端与所述上游引物甲部分相同，用于第一轮PCR的产物的扩增；
    所述index引物自5’端包括用于测序的区段A、用于区分样本的index序列和用于测序的区段B；
    所述下游引物组合乙中的引物具有所述区段B且与下游引物组合甲中检测相同目标靶点的引物形成巢式关系。
  13. 如权利要求12所述的方法，其特征在于：
    所述DNA样本中目标突变的分析方法为：将测序数据满足标准甲的DNA分子回溯到一个分子簇；将满足标准乙的分子簇标记为一对duplex分子簇；对某一突变来说，如果满足下述(a1)或(a2)，则该突变为来自原始DNA样本的真实突变：(a1)至少有一对duplex分子簇支持；(a2)至少有4个分子簇支持；标准甲即同时满足①、②和③；①DNA插入片段长度相同且除突变位点外序列一致；②随机标签序列相同；③锚定序列相同；标准乙即同时满足④和⑤；④DNA插入片段长度相同且除突变位点外序列一致；⑤分子簇两端的锚定序列相同但位置相反；
    所述DNA样本中甲基化的分析方法为：将测序数据满足标准丙的DNA分子标记为一个簇，分别计算片段末端为关注酶切位点的簇的数量，记录为未甲基化的片段；计算扩增片段达到或超过第一个酶切位点的全部簇的数量，记录为片段总数；根据两种片段数量计算对应区域的平均甲基化水平；区域的甲基化水平＝(1-未甲基化片段数/片段总数)×100％；标准丙即同时满足⑥、⑦和⑧；⑦随机标签序列相同；⑧锚定序列相同；⑨DNA插入片段长度相同且除突变位点外序列一致。
  14. 一种检测DNA样本中多种目标突变和/或甲基化的方法，包括如下步骤：
    (1)按照权利要求1至4任一所述的方法构建文库；
    (2)对步骤(1)的文库进行靶区域富集并进行测序，根据测序结果分析DNA样本中目标突变和/或甲基化的发生情况。
  15. 如权利要求14所述的方法，其特征在于：
    所述DNA样本中目标突变的分析方法为：将测序数据满足标准甲的DNA分子回溯到一个分子簇；将满足标准乙的分子簇标记为一对duplex分子簇；对某一突变来说，如果满足下述(a1)或(a2)，则该突变为来自原始DNA样本的真实突变：(a1)至少有一对duplex分子簇支持；(a2)至少有4个分子簇支持；标准甲即同时满足①、②和③；①DNA插入片段长度相同且除突变位点外序列一致；②随机标签序列相同；③锚定序列相同；标准乙即同时满足④和⑤；④DNA插入片段长度相同且除突变位点外序列一致；⑤分子簇两端的锚定序列相同但位置相反；所述DNA样本中甲基化的分析方法为：将测序数据满足标准丙的DNA分子标记为一个簇，分别计算片段末端为关注酶切位点的簇的数量，记录为未甲基化的片段；计算扩增片段达到或超过第一个酶切位点的全部簇的数量，记录为片段总数；根据两种片段数量计算对应区域的平均甲基化水平；区域的甲基化水平＝(1-未甲基化片段数/片段总数)×100％；标准丙即同时满足⑥、⑦和⑧；⑥随机标签序列相同；⑦锚定序列相同；⑧DNA插入片段长度相同且除突变位点外序列一致。
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